Method for the preparation of diols

ABSTRACT

The present invention concerns a new method for the biological preparation of a diol comprising culturing a microorganism genetically modified for the bioproduction of an aliphatic diol, wherein the microorganism comprises a metabolic pathway for the decarboxylation of a hydroxy-2-keto-aliphatic acid metabolite with an enzyme having a 2-keto acid decarboxylase activity, the product obtained from said decarboxylation step being further reduced into the corresponding aliphatic diol, and wherein the microorganism is genetically modified for the improved production of said hydroxy-2-keto-aliphatic acid metabolite. 
     The invention also concerns a modified microorganism for the production of an aliphatic diol.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Non-Provisional patent application claims priority to U.S. PatentApplication No. 61/361,459, filed Jul. 5, 2010. This Non-Provisionalpatent application is a continuation-in-part of PCT/EP2009/067994, filedDec. 29, 2009, which claims priority to European Application No.08173129.1, filed Dec. 31, 2008 and U.S. Patent Application No.61/141,699, filed Dec. 31, 2008. Each application cited is herebyincorporated by reference in its entirety.

BACKGROUND

1. Field of the Invention

The present invention concerns a new method for the biologicalpreparation of a diol comprising culturing a microorganism geneticallymodified for the bioproduction of an aliphatic diol, wherein themicroorganism comprises a metabolic pathway for the decarboxylation of ahydroxy-2-keto-aliphatic acid metabolite with two enzymes: an enzymehaving a 2-keto acid decarboxylase activity and an enzyme having ahydroxy aldehyde reductase activity. The invention also concerns amodified microorganism for the production of an aliphatic diol.

2. Description of Related Art

Fermentative production of diols by culturing microorganism producingsuch diols are known in the art, including fermentative production withmicroorganisms genetically modified for an improved production of thediols. Production of such diols is described, inter alia, in thefollowing documents: WO1996/035796, WO2001/012833, WO2004/033646, U.S.Pat. No. 7,267,972. In particular, production of 1,3-propanediol hasalready been described, involving vitamin B12-dependent enzymes, therebymaking the production process very expensive.

There is an ongoing need for alternative solutions of modifiedmicroorganisms, to either or both produce diols from renewable sourcesof carbon and have potential improvement in the production of the diols,particularly with vitamin B12-independent pathways. These technicalimprovements may be on the overall yield of product being produced basedon the energy necessary for such production and eventually, the level ofimpurities and by-products to be specifically controlled for isolationof the product and its marketing and further use.

SUMMARY OF THE INVENTION

The present invention concerns a microorganism genetically modified forthe bioproduction of an aliphatic diol, wherein the microorganismcomprises a metabolic pathway for the decarboxylation of ahydroxy-2-keto-aliphatic acid metabolite with an enzyme having a 2-ketoacid decarboxylase activity, the product obtained from saiddecarboxylation step being further reduced into the correspondingaliphatic diol with an enzyme having a hydroxy aldehyde reductaseactivity, and wherein the microorganism is genetically modified for theimproved production of said hydroxy-2-keto-aliphatic acid metabolite.

The microorganism of the invention is generally selected among the groupconsisting of a bacterium, yeast or a fungus. Preferentially, themicroorganism is selected among Enterobacteriaceae, Clostridiaceae,Bacillaceae, Streptomycetaceae and Corynebacteriaceae.

According to a first embodiment the microorganism comprises anendogenous gene coding for a 2-keto acid decarboxylase activity. It ispreferably selected among Saccharomyces cerevisiae (Pdc1, Pdc5, Pdc6,Aro10, Thi3); Lactococcus lactis (Kivd); Clostridium acetobutylicum(Pdc); Arabidopsis thaliana (Pdc2, Pdc3); Pichia stipitis (Pdc1, Pdc2,Aro10); Zymomonas mobilis (Pdc); Mycobacterium tuberculosis. Thismicroorganism having endogenous 2-keto acid decarboxylase activity canbe further modified to enhance expression of the endogenous gene codingfor the 2-keto acid decarboxylase.

According to another embodiment of the invention, the microorganism doesnot comprise an endogenous gene coding for a 2-keto acid decarboxylase.Such microorganism lacking endogenous 2-keto acid decarboxylase ispreferably selected among Escherichia coli or Corynebacterium glutamicumor Bacillus subtilis. For such microorganisms, the microorganism of theinvention comprises a heterologous gene coding for a 2-ketoaciddecarboxylase.

According to another embodiment the microorganism comprises anendogenous gene coding for a hydroxy aldehyde reductase activity. It ispreferably selected among Escherichia coli (yqhD, fucO, dkgA, dkgB);Saccharomyces cerevisiae (ADH1, ADH2, . . . ); and all organisms havingat least one enzyme having aldehyde reductase activity or alcoholdehydrogenase activity. This microorganism having endogenous hydroxyaldehyde reductase activity can be further modified to enhanceexpression of the endogenous gene coding for the hydroxy aldehydereductase.

The aliphatic diol produced with the microorganism of the invention isan aliphatic diol having a linear or branched alkyle chain comprisingfrom 2 to 6 carbon atoms, preferably 2, 3 or 4 carbon atoms.

In a preferred embodiment, the aliphatic diol is ethylene-glycol and thehydroxy-2-keto-aliphatic acid metabolite is hydroxypyruvate.

In another preferred embodiment, the aliphatic diol is 1,3-propanedioland the hydroxy-2-keto-aliphatic acid metabolite is4-hydroxy-2-ketobutyrate.

In another preferred embodiment, the aliphatic diol is 1,4-butanedioland the hydroxy-2-keto-aliphatic acid metabolite is5-hydroxy-2-ketopentanoate.

The present invention also concerns a method for the bioproduction of analiphatic diol, comprising the steps of

-   -   culturing a microorganism of the invention as described above        and below on an appropriate culture medium comprising a source        of carbon and    -   recovering the aliphatic diol from the culture medium.

According to preferred embodiment of the invention, the diol is furtherpurified.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT Microorganisms

The microorganism of the invention is a microorganism being geneticallymodified or genetically engineered. This means, according to the usualmeaning of these terms, that the microorganism of the invention is notfound in nature and is modified either by introduction or by deletion ofnew genetic elements. It can also be transformed by forcing thedevelopment and evolution of new metabolic pathways in combiningdirected mutagenesis and evolution under specific selection pressure(see for instance WO 2004/076659).

According to the invention, the term “microorganism” designates abacterium, yeast or a fungus. Preferentially, the microorganism isselected among Enterobacteriaceae, Clostridiaceae, Bacillaceae,Streptomycetaceae and Corynebacteriaceae. More preferentially themicroorganism is a species of Escherichia, Clostridium, Bacillus,Klebsiella, Pantoea, Salmonella or Corynebacterium. Even morepreferentially the microorganism is either the species Escherichia colior Corynebacterium glutamicum or Clostridium acetobutylicum or Bacillussubtilis.

A microorganism can express exogenous genes if these genes areintroduced into the microorganism with all the elements allowing theirexpression in the host microorganism. Transforming microorganisms withexogenous DNA is a routine task for the man skilled in the art.

Exogenous genes can be integrated into the host genome, or be expressedextrachromosomally by plasmids or vectors. Different types of plasmidsare known by the man skilled in the art, which differ with respect totheir origin of replication and their copy number in the cell.

Important elements for controlling the expression of genes arepromoters. In a preferred embodiment of the invention, genes may beexpressed using promoters with different strength, which may beinducible. These promoters may be homologous or heterologous. The manskilled in the art knows how to choose the promoters that are the mostconvenient, for example promoters Ptrc, Ptac, Plac or the lambdapromoter a are widely used.

In specific embodiments, endogenous genes can also be modified tomodulate their expression and/or activity, by introducing eithermutations in the coding sequence to modify the gene product or byintroducing heterologous sequences in addition or in replacement of theendogenous regulatory elements. Modulation of an endogenous gene can goboth ways: upregulating and/or enhancing the activity of the geneproduct on the one hand, or down regulating and/or lowering the activityof the endogenous gene product on the other hand.

The term ‘attenuation of a gene’ according to the invention denotes thepartial or complete suppression of the expression of a gene, which isthen said to be ‘attenuated’. This suppression of expression can beeither an inhibition of the expression of the gene, a deletion of all orpart of the promoter region necessary for the gene expression, adeletion in the coding region of the gene, or the replacement of thewild-type promoter by a weaker natural or synthetic promoter.Preferentially, the attenuation of a gene is essentially the completedeletion of that gene, which can be replaced by a selection marker genethat facilitates the identification, isolation and purification of thestrains according to the invention. A gene is inactivated preferentiallyby the technique of homologous recombination (Datsenko, K. A. & Wanner,B. L. (2000) “One-step inactivation of chromosomal genes in Escherichiacoli K-12 using PCR products”. Proc. Natl. Acad. Sci. USA 97:6640-6645).

In other embodiments of the invention, endogenous sequences may also beknocked out or deleted, to favour the new metabolic pathway.

All techniques for transforming the microorganisms, and regulatoryelements used for enhancing production of the protein of the inventionare well known in the art and available in the literature, includingapplicant's own patent applications on modification of biosynthesispathways in various microorganisms, including WO 2008/052973, WO2008/052595, WO 2008/040387, WO 2007/144346, WO 2007/141316, WO2007/077041, WO 2007/017710, WO 2006/082254, WO 2006/082252, WO2005/111202, WO 2005/073364, WO 2005/047498, WO 2004/076659, the contentof which is incorporated herein by reference.

Genes and Enzymatic Activities

In the description of the present invention, enzymatic activities arealso designated by reference to the genes coding for the enzymes havingsuch activity. Except mentioned otherwise, genes and proteins aregenerally identified using the denominations of genes from Escherichiacoli. However, use of these denominations has a more general meaningaccording to the invention and covers all the corresponding genes andproteins in other organisms, more particularly microorganisms,functional homologues, functional variants and functional fragments ofsaid genes and proteins.

Genes being identified in the present application are listed in FIG. 4with their accession number.

Using the references of the IUBMB Enzyme Nomenclature for knownenzymatic activities, those skilled in the art are able to determine thesame enzymatic activities in other organisms, bacterial strains, yeasts,fungi, etc. This routine work is advantageously done using consensussequences that can be determined by carrying out sequence alignmentswith proteins derived from other microorganisms.

Methods for the determination of the percentage of homology between twoprotein sequences are known from the man skilled in the art. Forexample, it can be made after alignment of the sequences by using thesoftware CLUSTALW available on the European Bioinformatics Institutewebsite with the default parameters indicated on the website. From thealignment, calculation of the percentage of identity can be made easilyby recording the number of identical residues at the same positioncompared to the total number of residues. Alternatively, automaticcalculation can be made by using for example the BLAST programsavailable on the National Center for Biotechnology Information websitewith the default parameters indicated on the website.

PFAM (protein families database of alignments and hidden Markov models;that can be used on the Wellcome Trust Sanger Institute website)represents a large collection of protein sequence alignments. Each PFAMmakes it possible to visualize multiple alignments, see protein domains,evaluate distribution among organisms, gain access to other databases,and visualize known protein structures.

COGs (clusters of orthologous groups of proteins; that can be used onthe National Center for Biotechnology Information website are obtainedby comparing protein sequences from 66 fully sequenced genomesrepresenting 30 major phylogenic lines. Each COG is defined from atleast three lines, which permits the identification of former conserveddomains.

A protein sharing homology with the cited protein may be obtained fromother microorganisms or may be a variant or a functional fragment of anatural protein.

The term “functional variant or functional fragment” means that theamino-acid sequence of the polypeptide may not be strictly limited tothe sequence observed in nature, but may contain additional amino-acids.The term “functional fragment” means that the sequence of thepolypeptide may include less amino-acid than the original sequence butstill enough amino-acids to confer the enzymatic activity of theoriginal sequence of reference. It is well known in the art that apolypeptide can be modified by substitution, insertion, deletion and/oraddition of one or more amino-acids while retaining its enzymaticactivity. For example, substitution of one amino-acid at a givenposition by a chemically equivalent amino-acid that does not affect thefunctional properties of a protein are common. For the purpose of thepresent invention, substitutions are defined as exchanges within one ofthe following groups:

-   -   Small aliphatic, non-polar or slightly polar residues: Ala, Ser,        Thr, Pro, Gly    -   Polar, negatively charged residues and their amides: Asp, Asn,        Glu, Gln    -   Polar, positively charged residues: H is, Arg, Lys    -   Large aliphatic, non-polar residues: Met, Leu, Ile, Val, Cys    -   Large aromatic residues: Phe, Tyr, Trp.

Changes that result in the substitution of one negatively chargedresidue for another (such as glutamic acid for aspartic acid) or onepositively charged residue for another (such as lysine for arginine) canbe expected to produce a functionally equivalent product.

The positions where the amino-acids are modified and the number ofamino-acids subject to modification in the amino-acid sequence are notparticularly limited. The man skilled in the art is able to recognizethe modifications that can be introduced without affecting the activityof the protein. For example, modifications in the N- or C-terminalportion of a protein may be expected not to alter the activity of aprotein under certain circumstances.

The term “variant” refers to polypeptides submitted to modificationssuch as defined above while still retaining the original enzymaticactivity.

The terms “encoding” or “coding” refer to the process by which apolynucleotide, through the mechanisms of transcription and translation,produces an amino-acid sequence. This process is allowed by the geneticcode, which is the relation between the sequence of bases in DNA and thesequence of amino-acids in proteins. One major feature of the geneticcode is that it is degenerate, meaning that one amino-acid can be codedby more than one triplet of bases (one “codon”). The direct consequenceis that the same amino-acid sequence can be encoded by differentpolynucleotides. It is well known from the man skilled in the art thatthe use of codons can vary according to the organisms. Among the codonscoding for the same amino-acid, some can be used preferentially by agiven microorganism. It can thus be of interest to design apolynucleotide adapted to the codon usage of a particular microorganismin order to optimize the expression of the corresponding protein in thisorganism.

In some instance, genes or enzymes may be designated by the name of theactivity. In some other instances, the designation by “activity” maymean a combination of two or more enzymes having in combination thedesired activity. In such case, each enzyme in the combination may beencoded by distinct genes under control of different regulatory elementsor a combination of genes under control of the same operon.

Genes coding for a 2-keto acid decarboxylase activity are well known inthe art, including Pdc genes from various species, and more particularlythe Pdc1, Pdc5, Pdc6, Aro10 and Thi3 genes from Saccharomycescerevisiae, kivD gene from Lactococcus lactis; pdc gene from Clostridiumacetobutylicum; Pdc2 and Pdc3 genes from Arabidopsis thaliana; Pdc1,Pdc2 and Aro10 genes from Pichia stipitis; pdc gene from Zymomonasmobilis. The first subunit of the 2-ketoglutarate decarboxylase complex,encoded by the gene sucA from Escherichia coli, also possesses 2-ketoacid decarboxylase activity, as well as the enzyme encoded by the genedxs of Escherichia coli. Functional homologues, functional variants andfunctional fragments of said genes and proteins are encompassed by thedefinition.

Genes coding for a hydroxy aldehyde reductase activity are also wellknown in the art, including the yqhD, fucO, dkgA, dkgB genes fromEscherichia coli and the ADH1 and ADH2 genes from Saccharomycescerevisiae. Functional homologues, functional variants and functionalfragments of said genes and proteins are encompassed by the definition.

Fermentative Production

The present invention also concerns the fermentative production of analiphatic diol, comprising the steps of

-   -   culturing a microorganism on an appropriate culture medium        comprising a source of carbon and    -   recovering the aliphatic diol from the culture medium.

The fermentation is generally conducted in fermenters with anappropriate culture medium adapted to the microorganism being used,containing at least one simple carbon source, and if necessaryco-substrates.

An ‘appropriate culture medium’ designates a medium (e.g., a sterile,liquid media) comprising nutrients essential or beneficial to themaintenance and/or growth of the cell such as carbon sources or carbonsubstrate, nitrogen sources, for example, peptone, yeast extracts, meatextracts, malt extracts, urea, ammonium sulfate, ammonium chloride,ammonium nitrate and ammonium phosphate; phosphorus sources, forexample, monopotassium phosphate or dipotassium phosphate; traceelements (e.g., metal salts), for example magnesium salts, cobalt saltsand/or manganese salts; as well as growth factors such as amino acids,vitamins, growth promoters, and the like.

As an example of known culture mediums for E. coli, the culture mediumcan be of identical or similar composition to an M9 medium (Anderson,1946, Proc. Natl. Acad. Sci. USA 32:120-128), an M63 medium (Miller,1992; A Short Course in Bacterial Genetics: A Laboratory Manual andHandbook for Escherichia coli and Related Bacteria, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) or a medium such as definedby Schaefer et al. (1999, Anal. Biochem. 270: 88-96).

As another example of a culture medium for C. glutamicum, the culturemedium can be of identical or similar composition to BMCG medium (Lieblet al., 1989, Appl. Microbiol. Biotechnol. 32: 205-210) or to a mediumsuch as described by Riedel et al. (2001, J. Mol. Microbiol. Biotechnol.3: 573-583).

The term ‘carbon source’ or ‘carbon substrate’ or ‘source of carbon’according to the present invention denotes any source of carbon that canbe used by those skilled in the art to support the normal growth of amicro-organism, including hexoses (such as glucose, galactose orlactose), pentoses, monosaccharides, disaccharides, oligosaccharides(such as sucrose, cellobiose or maltose), molasses, starch or itsderivatives, hemicelluloses, glycerol and combinations thereof. Anespecially preferred simple carbon source is glucose. Another preferredsimple carbon source is sucrose.

In some embodiments of the invention, the culture medium comprises acarbon source being a by-product of another process using biomass asstarting material, or eventually, the product of mechanical and/orchemical and/or enzymatic, and in such instance in vitro or in vivo,degradation of biomass, such as degradation of cellulose.

The microorganism of the invention is advantageously elected and/ormodified to use the source of carbon as sole source of carbon to grow onthe culture medium.

Microorganisms selected to grow on a specific source of carbon are knownin the art, as well as modifications to be introduced in a microorganismto allow it to grow on said specific source of carbon.

Those skilled in the art are able to define the culture conditions forthe microorganisms according to the invention. In particular thebacteria are fermented at a temperature between 20° C. and 55° C.,preferentially between 25° C. and 40° C., and more specifically about30° C. for C. glutamicum and about 37° C. for E. coli.

Recovering the aliphatic diol from the culture medium is a routine taskfor a man skilled in the art.

In one aspect of the invention, the recovered aliphatic diol is furtherpurified.

Methods for recovering a diol from a culture medium and its purificationare known in the art and disclosed, inter alia in the followingdocuments: PCT/EP2008/063287 filed on Oct. 3, 2008 and PCT/EP2007/063068filed on Nov. 30, 2007.

Specific Embodiments

Other embodiments of the invention will be described below. Themicroorganisms are modified both to favor the production of thehydroxy-2-keto-aliphatic acid metabolite and the transformation into thecorresponding aliphatic diol of the product obtained from thedecarboxylation step of the same hydroxy-2-keto-aliphatic acidmetabolite.

The description below is made by reference to E. coli, whichmicroorganism is lacking endogenous 2-keto acid decarboxylase activity.Therefore, a heterologous gene coding for said activity is introducedinto the microorganism.

Modifications of the microorganism to optimise the pathway for producingthe hydroxy-2-keto-aliphatic acid metabolite and to transform theproduct obtained from the decarboxylation step of the samehydroxy-2-keto-aliphatic acid metabolite into the aliphatic diol is alsomade based on the known metabolic pathways and endogenous genes of E.coli. However, the man skilled in the art can use similar strategies tointroduce or delete corresponding genes in other microorganisms withknown genes and pathways.

I. Preparation of Ethylene Glycol

The biosynthesis pathway for the production of ethylene glycol accordingto the invention comprises three enzymatic reactions starting withtransformation of the 3-phosphohydroxypyruvate precursor (precursor forserine). First a phosphatase activity allows conversion ofphosphohydroxypyruvate into hydroxypyruvate. Hydroxypyruvate is thentransformed into glycolaldehyde with a 2-keto acid decarboxylaseactivity. Finally, a hydroxy aldehyde reductase activity allows theconversion of glycolaldehyde into ethylene glycol. Another pathway forthe production of ethylene glycol starts from L-serine as precursor.First a transaminase or an amino acid oxidase activity allows conversionof serine into hydroxypyruvate. The next two steps are similar to thefirst pathway described above.

The global biosynthesis pathway is represented in FIG. 1.

The present invention provides a method for the fermentative productionof ethylene glycol, its derivatives or precursors, comprising: culturinga microorganism, particularly a bacterium, in an appropriate culturemedium comprising a source of carbon and recovering ethylene glycol fromthe culture medium.

In a preferred embodiment, the method is performed with a microorganism,particularly a bacterium, which contains at least one gene encoding apolypeptide with 2-keto acid decarboxylase activity and one geneencoding a polypeptide with hydroxy aldehyde reductase activity. Thosegenes can be exogenous or endogenous, and can be expressed chromosomallyor extrachromosomally.

In a further embodiment of the invention, the method is performed with amicroorganism, particularly a bacterium, in which the availability ofthe intermediate 3-phosphoglycerate is increased. Preferably, theincrease is achieved by attenuating the level of expression of genesencoding phosphoglycerate mutases, in particular one or both genes gpmAand pgmI. This can be done by replacing the wild-type promoter of thesegenes by a weaker promoter, or by the use of an element destabilizingthe corresponding messenger RNA or the protein. If needed, completeattenuation of the genes can also be achieved by the deletion of thecorresponding DNA sequences. The invention is also related to themicroorganism used in this particular embodiment of the invention, i.e.a microorganism, particularly a bacterium presenting an increasedavailability of 3-phosphoglycerate, in particular a microorganism,preferentially a bacterium, in which the expression of the genes codingfor phosphoglycerate mutases is attenuated, preferably the expression ofone or both genes gpmA and pgmI.

In another embodiment, the method is performed with a microorganism,particularly a bacterium, in which flux into the serine biosynthesispathway is stimulated. This can be achieved by increasing the level ofexpression of 3-phosphoglycerate dehydrogenase and/or phosphoserineaminotransferase, encoded by the serA and serC genes, respectively.Increasing the level of expression of the 3-phosphoglyceratedehydrogenase and/or phosphoserine aminotransferase can be accomplishedby introducing artificial promoters that drive the expression of theserA and/or serC genes, by increasing the number of copies in the cellor by introducing mutations into the serA and/or serC genes thatincrease the activity of the corresponding proteins. The expression ofthe serA gene can also be increased by replacing the wild type lrp gene(encoding the leucine-responsive regulatory protein) by an lrp mutatedallele (such as the lrp-1 allele corresponding to a GLU114ASPsubstitution in the lrp protein) leading to the constitutive activationof the transcription of the serA gene. The invention is also related tothe microorganism, particularly a bacterium, used in this particularembodiment of the invention.

In a particular embodiment of the invention mutations can be introducedinto the serA gene that reduce the sensitivity of the SerA protein tothe feed-back inhibitor serine (feed-back desensitized alleles) and thuspermit an increased activity in the presence of serine. Examples ofdesensitized alleles, i.e. feed-back insensitive alleles, have beendescribed in EP 0 931 833 (Ajinomoto) or EP 0 620 853 (Wacker).

In another embodiment the method is performed with a microorganism,particularly a bacterium, in which flux into the hydroxypyruvatebiosynthesis pathway is stimulated. This result can be achieved byincreasing the level of expression of serine transaminase or serineoxidase (for the pathway starting from serine as precursor), or byincreasing the expression of 3-phosphohydroxypyruvate phosphatase.Increasing the level of expression of serine oxidase can be accomplishedby introducing and overexpressing the gene coding for L-amino acidoxidase from R. opacus, or by introducing mutations into the gene thatincrease the activity of the corresponding protein. An increase in theexpression of serine transaminase can be accomplished by introducingartificial promoters that drive the expression of the serC gene of E.coli, by increasing the number of copies in the cell or by introducingmutations into the serC gene that increase the activity of thecorresponding protein. An increase of the expression of3-phosphohydroxypyruvate phosphatase can be accomplished by introducingartificial promoters that drive the expression of the yeaB gene or serBgene of E. coli, by increasing the number of copies in the cell or byintroducing mutations into the yeaB gene or the serB gene that increasethe activity of the corresponding proteins. An increase of theexpression of 3-phosphohydroxypyruvate phosphatase can also beaccomplished by introducing and overexpressing the gene GPP2 from S.cerevisiae, or by introducing mutations into the GPP2 gene that increasethe activity of the corresponding protein. The invention is also relatedto the microorganism, particularly a bacterium, used in this particularembodiment of the invention.

In a further embodiment of the invention, the microorganism,particularly a bacterium, is modified to present an attenuated level ofserine conversion to other compounds than ethylene glycol. This resultmay be achieved by attenuating the level of serine consuming enzymeslike serine deaminases (encoded by sdaA and sdaB and tdcG), serinetransacetylase (encoded by cysE), tryptophan synthase (encoded by trpAB)or serine hydroxymethyltransferase (encoded by glyA). These genes can beattenuated by replacing the natural promoter by a lower strengthpromoter or by elements destabilizing the corresponding messenger RNA orthe protein. If needed, complete attenuation of the gene can also beachieved by a deletion of the corresponding DNA sequence. The inventionis also related to the microorganism, particularly a bacterium, used inthis particular embodiment of the invention.

In a further embodiment of the invention, the microorganism,particularly a bacterium, is modified to present an attenuated level ofhydroxypyruvate conversion to other compounds than glycolaldehyde. Thisresult may be achieved by attenuating the level of hydroxypyruvateconsuming enzymes like hydroxypyruvate reductase (encoded by ghrA) orhydroxypyruvate isomerase (encoded by hyi). These genes can beattenuated by replacing the natural promoter by a lower strengthpromoter or by elements destabilizing the corresponding messenger RNA orthe protein. If needed, complete attenuation of the gene can also beachieved by a deletion of the corresponding DNA sequence. The inventionis also related to the microorganism, particularly a bacterium, used inthis particular embodiment of the invention.

In a further embodiment of the invention, the microorganism,particularly a bacterium, is modified to present an attenuated level ofglycolaldehyde conversion to other compounds than ethylene glycol Thismay be achieved by attenuating the level of glycolaldehyde consumingenzymes like hydroxythreonine aldolase (encoded by ltaE) orglycolaldehyde dehydrogenase (encoded by aldA, aldB). Attenuation ofthese genes can be done by replacing the natural promoter by a lowerstrength promoter or by elements destabilizing the correspondingmessenger RNA or the protein. If needed, complete attenuation of thegene can also be achieved by a deletion of the corresponding DNAsequence. The invention is also related to the microorganism,particularly a bacterium, used in this particular embodiment of theinvention.

In one aspect of the invention, the efficiency of sugar import isincreased, either by using a sugar import system not relying onphosphoenolpyruvate (PEP) as phosphordonor like galP that is known totransport glucose, or by providing more phosphoenolpyruvate (PEP) to thesugar-phosphotransferase system. Various means exist that may be used toincrease the availability of PEP in a microorganism. In particular, thiscan be accomplished by attenuating the reaction PEP→pyruvate.Preferentially, at least one gene selected among pykA and pykF, encodingpyruvate kinase, is attenuated in said strain in order to obtain thisresult. Another way to increase the availability of PEP is to favour thereaction pyruvate→PEP. This can be accomplished by increasing theactivity of phosphoenolpyruvate synthase which catalyzes the abovereaction. This enzyme is encoded by the ppsA gene. Therefore, in themicroorganism, the expression of the ppsA gene is preferentiallyincreased. Both modifications can be present in the microorganismsimultaneously.

II. Preparation of 1,3-propanediol

The biosynthesis pathway for the production of 1,3-propanediol accordingto the invention comprises three enzymatic reactions starting withtransformation of the L-homoserine precursor (obtained fromL-aspartate). First a homoserine transaminase or a homoserine oxidaseactivity allows conversion of L-homoserine into4-hydroxy-2-ketobutyrate. A second 2-keto acid decarboxylase activityallows conversion of 4-hydroxy-2-ketobutyrate into3-hydroxypropionaldehyde. 3-hydroxypropionaldehyde is then convertedinto 1,3-propanediol with a hydroxy aldehyde reductase activity.

The global biosynthesis pathway is represented in FIG. 2.

The present invention provides a method for the fermentative productionof 1,3-propanediol, its derivatives or precursors, comprising: culturinga microorganism, particularly a bacterium, in an appropriate culturemedium comprising a source of carbon and recovering 1,3-propanediol fromthe culture medium.

In a preferred embodiment, the method is performed with a microorganism,particularly a bacterium, which contains at least one gene encoding apolypeptide with 2-keto acid decarboxylase activity and one geneencoding a polypeptide with hydroxy aldehyde reductase activity. Thosegenes can be exogenous or endogenous, and can be expressed chromosomallyor extrachromosomally.

In another embodiment, the method is performed with a microorganism,particularly a bacterium, whose flux in the oxaloacetate biosynthesispathway is stimulated; this result can be achieved by increasing thelevel of expression of phosphoenolpyruvate carboxylase, encoded by theppc gene. Increasing the expression of phosphoenolpyruvate carboxylasecan be accomplished by introducing artificial promoters that drive theexpression of the ppc gene, by increasing the number of copies in thecell or by introducing mutations into the ppc gene that increase theactivity of the corresponding protein. The availability of theintermediate product oxaloacetate can also be increased by attenuatingthe level of expression of genes coding for phosphoenolpyruvatecarboxykinase and/or malic enzymes, encoded by the pckA and/or maeAand/or maeB genes, respectively. This can be done by replacing thewild-type promoter of these genes by a weaker promoter, or by the use ofan element destabilizing the corresponding messenger RNA or the protein.If needed, complete attenuation of the genes can also be achieved by thedeletion of the corresponding DNA sequences. The invention is alsorelated to the microorganism, particularly a bacterium, used in thisparticular embodiment of the invention, i.e. a microorganism,particularly a bacterium, presenting an increased availability of theoxaloacetate.

In another embodiment, the method is performed with a microorganism,particularly a bacterium, in which flux into the homoserine biosynthesispathway is stimulated. This can be achieved by increasing the expressionof aspartokinase and homoserine dehydrogenase and/or aspartatesemialdehyde dehydrogenase, encoded by the thrA and asd genes,respectively. Increasing the expression of aspartokinase and homoserinedehydrogenase and/or aspartate semialdehyde dehydrogenase can beaccomplished by introducing artificial promoters that drive theexpression of the thrA and/or asd genes, by increasing the number ofcopies in the cell or by introducing mutations into the thrA and/or asdgenes that increase the activity of the corresponding proteins. Theinvention is also related to the microorganism, particularly abacterium, used in this particular embodiment of the invention.

In a particular embodiment of the invention mutations can be introducedinto the thrA gene that reduce its sensitivity to the feed-backinhibitor threonine (feed-back desensitized alleles) and thus permit anincreased activity in the presence of threonine.

In another embodiment, the method is performed with a microorganism,particularly a bacterium, in which flux into the4-hydroxy-2-ketobutyrate biosynthesis pathway is stimulated. This resultcan be achieved by increasing the expression of homoserine transaminaseor homoserine oxidase. Increasing the expression of homoserine oxidasecan be accomplished by introducing and overexpressing the gene codingfor L-amino acid oxidase from R. opacus, or by introducing mutationsinto the gene that increase the activity of the corresponding protein.Increasing the level of expression of homoserine transaminase can beaccomplished by introducing artificial promoters that drive theexpression of the serC gene of E. coli, by increasing the number ofcopies in the cell or by introducing mutations into the serC gene thatincrease the activity of the corresponding protein. The invention isalso related to the microorganism, particularly a bacterium, used inthis particular embodiment of the invention.

In a further embodiment of the invention, the microorganism,particularly a bacterium, is modified to present an attenuated level ofhomoserine conversion to other compounds than 1,3-propanediol. Thisresult may be achieved by attenuating the level of homoserine consumingenzymes like homoserine kinase and threonine synthase (encoded by thrBand thrC), homoserine O-transsuccinylase (encoded by metA) ordihydrodipicolinate synthase (encoded by dapA). These genes can beattenuated by replacing the natural promoter by a weaker promoter or byelements destabilizing the corresponding messenger RNA or protein. Ifneeded, complete attenuation of the gene can also be achieved by thedeletion of the corresponding DNA sequence. The invention is alsorelated to the microorganism, particularly a bacterium, used in thisparticular embodiment of the invention.

In a further embodiment of the invention, the microorganism,particularly a bacterium, is modified to present an attenuated level of3-hydroxypropionaldehyde conversion to other compounds than1,3-propanediol. This may be achieved by attenuating the level of3-hydroxypropionaldehyde consuming enzymes like 3-hydroxypropionaldehydedehydrogenase (encoded by aldA, aldB). These genes can be attenuated byreplacing the natural promoter by a weaker promoter or by elementsdestabilizing the corresponding messenger RNA or the protein. If needed,complete attenuation of the gene can also be achieved by the deletion ofthe corresponding DNA sequence. The invention is also related to themicroorganism, particularly a bacterium, used in this particularembodiment of the invention.

In one aspect of the invention, the efficiency of the sugar import isincreased, either by using a sugar import system not relying onphosphoenolpyruvate (PEP) as phosphordonor such as the one encoded bygalP that is known to transport glucose, or by providing morephosphoenolpyruvate (PEP) to the sugar-phosphotransferase system.Various means exist that may be used to increase the availability of PEPin a microorganism. In particular, this may be accomplished byattenuating the reaction PEP→pyruvate. Preferentially, at least one geneselected among pykA and pykF, encoding pyruvate kinase, is attenuated insaid strain in order to obtain this result. Another way to increase theavailability of PEP is to favour the reaction pyruvate→PEP. This can beaccomplished by increasing the activity of phosphoenolpyruvate synthasewhich catalyzes the above reaction. This enzyme is encoded by the ppsAgene. Therefore, in the microorganism, the expression of the ppsA geneis preferentially increased. Both modifications can be present in themicroorganism simultaneously.

III. Preparation of 1,4-butanediol

The biosynthesis pathway for the production of 1,4-butanediol accordingto the invention comprises five enzymatic reactions starting withtransformation of the 2-ketoglutarate precursor (metabolite of the Krebscycle).

A first activity, 4-oxoglutaryl-CoA synthetase, allows conversion of2-ketoglutarate into 4-oxoglutaryl-CoA. This compound is then convertedinto 5-hydroxy-2-ketopentanoate with the combinations of two activities,first aldehyde dehydrogenase then alcohol dehydrogenase both encoded bythe gene adhE2 of Clostridium acetobutylicum or adhE of Escherichiacoli. 2-keto acid decarboxylase activity allows then the conversion of5-hydroxy-2-oxopentanoate into 4-hydroxybutyraldehyde, which is furtherconverted into 1,4-butanediol relying on hydroxy aldehyde reductaseactivity.

The global biosynthesis pathway is represented on FIG. 3.

The present invention provides a method for the fermentative productionof 1,4-butanediol, its derivatives or precursors, comprising: culturinga microorganism, particularly a bacterium, in an appropriate culturemedium comprising a source of carbon and recovering 1,4-butanediol fromthe culture medium.

In a preferred embodiment, the method is performed with a microorganism,particularly a bacterium, which contains at least one gene encoding apolypeptide with 2-keto acid decarboxylase activity and one geneencoding a polypeptide with hydroxy aldehyde reductase activity. Thosegenes can be exogenous or endogenous, and can be expressed chromosomallyor extrachromosomally.

In another embodiment, the method is performed with a microorganism,particularly a bacterium, in which flux into the oxaloacetatebiosynthesis pathway is stimulated (entry of the Krebs cycle). This canbe achieved by increasing the expression of phosphoenolpyruvatecarboxylase, encoded by the ppc gene. Increasing the expression ofphosphoenolpyruvate carboxylase can be accomplished by introducingartificial promoters that drive the expression of the ppc gene, byincreasing the number of copies in the cell or by introducing mutationsinto the ppc gene that increase the activity of the correspondingprotein. Availability of the intermediate product oxaloacetate can alsobe increased by attenuating the level of expression of genes coding forphosphoenolpyruvate carboxykinase and/or malic enzymes, encoded by thepckA and/or maeA and/or maeB genes, respectively. This can be done byreplacing the wild-type promoter of these genes by a weaker promoter, orby the use of an element destabilizing the corresponding messenger RNAor the protein. If needed, complete attenuation of the genes can also beachieved by the deletion of the corresponding DNA sequences. Theinvention is also related to the microorganism, particularly abacterium, used in this particular embodiment of the invention, i.e. amicroorganism, particularly a bacterium, presenting an increasedavailability of 2-ketoglutarate.

In another embodiment, the method is performed with a microorganism,particularly a bacterium, in which flux into the 2-ketoglutaratebiosynthesis pathway is stimulated. This can be achieved by increasingthe expression of citrate synthase and/or isocitrate dehydrogenase,encoded by the gltA and icd genes, respectively. Increasing theexpression of citrate synthase and/or isocitrate dehydrogenase can beaccomplished by introducing artificial promoters that drive theexpression of the gltA and/or icd genes, by increasing the number ofcopies in the cell or by introducing mutations into the gltA and/or icdgenes that increase the activity of the corresponding proteins.Isocitrate dehydrogenase activity is modulated by phosphorylation ordephosphorylation, reactions that are catalyzed by AceK. Phosphorylationreduces the activity of Icd and dephosphorylation reactivates the Icdenzyme. The activity of the Icd enzyme may therefore also be controlledby introducing mutant aceK genes that have reduced kinase activity orincreased phosphatase activity compared to the wild type AceK enzyme.The level of AceK activity can also be decreased by attenuating theexpression of the aceK gene. This can be done by replacing the wild-typepromoter of the gene by a weaker promoter, or by the use of an elementdestabilizing the corresponding messenger RNA or the protein. If needed,complete attenuation of the gene can also be achieved by the deletion ofthe corresponding DNA sequence. Availability of the intermediate2-ketoglutarate can also be increased by attenuating the expression ofgenes coding for 2-ketoglutarate decarboxylase or succinyl-CoAsynthetase and/or isocitrate lyase or malate synthase, encoded by thesucAB or sucCD and/or aceA or aceB genes, respectively. This can be doneby replacing the wild-type promoter of these genes by a weaker promoter,or by the use of an element destabilizing the corresponding messengerRNA or the protein. The flux in the Krebs cycle can also be increased byalleviating the repression of ArcA (encoded by the arcA gene) thatrepresses Krebs cycle encoding genes. The invention is also related tothe microorganism, particularly a bacterium, used in this particularembodiment of the invention, i.e. a microorganism, particularly abacterium, presenting an increased availability of the 2-ketoglutarate.

In another embodiment, the method is performed with a microorganism,particularly a bacterium, in which flux into the5-hydroxy-2-ketopentanoate biosynthesis pathway is stimulated. This canbe achieved by increasing the expression of the 4-oxoglutaryl-CoAsynthetase (AMP-forming, such as encoded by the prpE gene, orADP-forming such as encoded by the sucC and sucD genes) and/or thealdehyde reductase/alcohol dehydrogenase. The invention is also relatedto the microorganism, particularly a bacterium, used in this particularembodiment of the invention.

In a further embodiment of the invention, the microorganism,particularly a bacterium, is modified to present an attenuated level of2-ketoglutarate conversion to other compounds than 1,4-butanediol; Thismay be achieved by attenuating the level of 2-ketoglutarate consumingenzymes like glutamate dehydrogenase or glutamate synthase (encoded bygdhA and gltB). These genes can be attenuated by replacing the naturalpromoter by a weaker promoter or by elements destabilizing thecorresponding messenger RNA or the protein. If needed, completeattenuation of the gene can also be achieved by the deletion of thecorresponding DNA sequence. The invention is also related to themicroorganism, particularly a bacterium, used in this particularembodiment of the invention.

In a further embodiment of the invention, the microorganism,particularly a bacterium, is modified to present an attenuated level of4-hydroxybutyraldehyde conversion to other compounds than1,4-butanediol. This may be achieved by attenuating the level of4-hydroxybutyraldehyde consuming enzymes like 4-hydroxybutyraldehydedehydrogenase (encoded by aldA, aldB). These genes can be attenuated byreplacing the natural promoter by a weaker promoter or by elementsdestabilizing the corresponding messenger RNA or the protein. If needed,complete attenuation of the gene can also be achieved by the deletion ofthe corresponding DNA sequence. The invention is also related to themicroorganism, particularly a bacterium, used in this particularembodiment of the invention.

In a further embodiment of the invention, the microorganism,particularly a bacterium, is modified in order to produce 1,4-butanediolin anaerobic conditions. To achieve such capacity, in saidmicroorganism, the PTS-sugar transport system is deleted in order tometabolize sugar via a permease/kinase transport system. In order toproduce enough ATP for growth of the microorganism under anaerobicconditions, oxaloacetate must be produced from phosphoenolpyruvate viaphosphoenolpyruvate carboxykinase activity encoded by the pckA gene inE. coli. This generates one mole of ATP per mole of oxaloacetateproduced. In a similar manner, the conversion of 2-oxoglutarate into4-oxoglutaryl-CoA must be achieved by 4-oxoglutaryl-CoA synthetase. TheADP-forming activity, such as encoded by the sucC and sucD genes in E.coli, consumes only one mole of ATP per mole of 4-oxoglutaryl-CoAproduced. The described metabolic pathway to produce 1,4-butanediol fromD-glucose is particularly adapted for anaerobic growth conditions. Theglobal reaction balance of such pathway is:D-glucose+ADP+Pi→1,4-butanediol+formate+CO2+ATP+H2O. In a furtherembodiment of the invention, the microorganism, particularly abacterium, is modified to present an attenuated level of acetyl-CoAconversion to other compounds than 1,4-butanediol; this result may beachieved by attenuating the level of acetyl-CoA consuming enzymes likeacetate kinase and phosphate acetyltransferase (encoded by ackA andpta). Attenuation of these genes can be done by replacing the naturalpromoter by a lower strength promoter or by element destabilizing thecorresponding messenger RNA or the protein. If needed, completeattenuation of the gene can also be achieved by a deletion of thecorresponding DNA sequence. The invention is also related to themicroorganism, particularly a bacterium, used in this particularembodiment of the invention.

DRAWINGS

FIG. 1. Biosynthesis pathway of ethylene glycol

FIG. 2. Biosynthesis pathway of 1,3-propanediol

FIG. 3. Biosynthesis pathway of 1,4-butanediol

FIG. 4. List of genes identified in the present application.

EXAMPLES Example 1 Construction of Strains Expressing a 2-Keto AcidDecarboxylase Encoding Gene and a Hydroxy Aldehyde Reductase EncodingGene: MG1655

(pME101-kivDll-yqhD-TT07)

1.1 Construction of the Plasmid pM-Ptrc01-kivDll-TT07 for theOverexpression of kivD of Lactococcus lactis EncodingAlpha-Keto-Isovalerate Decarboxylase:

A synthetic gene of the Lactococcus lactis kivD coding for thealpha-keto-isovalerate decarboxylase was prepared by Geneart (Germany).The codon usage and GC content of the gene was adapted to Escherichiacoli according to the supplier matrix. Expression of the synthetic genewas driven by a constitutive Ptrc promoter. A transcriptional terminatorwas added downstream of the gene. The construct was cloned intosupplier's pM vectors and verified by sequencing. If necessary, thesynthetic gene was cloned into the pME101 vector (This plasmid isderived from plasmid pCL1920 (Lerner & Inouye, 1990, NAR 18, 15 p 4631))before transforming an E. coli strain.

Ptrc01-kivDll-TT07:

restriction sites (BamHI, HindIII, EcoRV) (SEQ ID NO 1):ggatccatgcaagcttatgcgatatc Ptrc01 promoter (SEQ ID NO 2):gagctgttgacaattaatcatccggctcgtataatgtgtggaataa ggaggtataackivDll gene sequence optimized for E. coli (CAG34226.) (SEQ ID NO 3):atgtataccgtgggtgattatctgctggatcgtctgcatgaactgggcattgaagaaattttcggcgttccgggtgattataatctgcagtttctggatcagattattagccataaagatatgaaatgggtgggtaatgccaatgaactgaatgcaagctatatggcagatggttatgcccgtaccaaaaaagcagcagcatttctgaccacctttggtgttggtgaactgagcgcagttaatggtctggctggtagctatgcagaaaatctgccggttgttgaaattgttggtagcccgaccagcaaagttcagaatgaaggcaaatttgtgcatcataccctggccgatggtgattttaaacatttcatgaaaatgcatgaaccggttaccgcagcacgtaccctgctgaccgcagaaaatgcaaccgttgaaattgatcgtgttctgagcgcactgctgaaagaacgtaaaccggtgtatattaatctgccggtggatgttgcagcagcaaaagcagaaaaaccgagcctgccgctgaaaaaagaaaatagcaccagcaataccagcgatcaggaaattctgaataaaattcaggaatccctgaaaaacgccaaaaaaccgattgtgattaccggtcatgaaattattagctttggcctggaaaaaaccgttacccagtttattagcaaaaccaaactgccgattaccaccctgaattttggtaaaagcagcgttgatgaagcactgccgagctttctgggtatttataatggcaccctgagcgaaccgaatctgaaagaatttgtggaaagcgcagatttcattctgatgctgggtgttaaactgaccgatagctctaccggtgcatttacccatcatctgaatgaaaacaaaatgattagcctgaatattgatgaaggcaaaatttttaatgaacgcattcagaattttgattttgaaagcctgattagcagcctgctggatctgagcgaaatcgaatataaaggcaaatatattgataaaaaacaggaagattttgttccgagcaatgcactgctgtctcaggatcgtctgtggcaggcagttgaaaatctgacccagagcaatgaaaccattgttgcagaacagggcaccagcttttttggtgcaagcagcatttttctgaaaagcaaaagccattttattggtcagccgctgtggggtagcattggttatacctttccggcagcactgggtagccagattgcagataaagaaagccgtcatctgctgtttattggtgatggtagcctgcagctgaccgttcaggaactgggtctggccattcgcgaaaaaattaatccgatttgctttattatcaataatgatggctataccgtggaacgtgaaattcatggtccgaatcagagctataatgatattccgatgtggaattatagcaaactgccggaatcttttggtgcaaccgaagatcgtgttgtgagcaaaattgtgcgcaccgaaaatgaatttgtgagcgtgatgaaagaagcacaggcagatccgaatcgtatgtattggattgaactgattctggccaaagaaggtgcaccgaaagttctgaaaaaaatgggcaaactgttcgccgaacagaataaaagctaaterminator sequence T7Te (ref: HarringtonK. J., Laughlin R. B. and Liang S. Proc NatlAcad Sci USA. 2001 Apr. 24; 98(9): 5019-24.) (SEQ ID NO 4):attacgtagaAGATCTtcctggctcaccttcgggtgggcctttctgrestriction sites (SmaI, BamHI, EcoRI) (SEQ ID NO 5):ccccgggatgcggatccatgcgaattc

For the expression from a low copy vector the pME101 plasmid wasconstructed as follows. The pCL1920 plasmid was PCR amplified using theoligonucleotides PME101F and PME101R and the BstZ17I-XmnI fragment fromthe vector pTRC99A harboring the lad gene and the Ptrc promoter wasinserted into the amplified vector. The resulting vector was restrictedby NcoI and BamHI and the vector harboring the kivDll gene wasrestricted by AflIII and BamHI. The kivDll containing fragment was thencloned into the vector pME101: The resulting plasmid was namedpME101-kivDll-TT07

PME101F (SEQ ID NO 6): ccgacagtaa gacgggtaag cctg PME101R (SEQ ID NO 7):agcttagtaa agccctcgct ag

1.2 Construction of a Plasmid pME101-kivDll-yqhD-TT07 for theOverexpression of kivD of Lactococcus lactis EncodingAlpha-Ketoisovalerate Decarboxylase and yqhD of Escherichia ColiEncoding Methylglyoxal Reductase

The pME101 vector and the vector harboring the kivDll genes wererestricted by SnaBI and BglII and the yqhD containing fragment wascloned into the vector pME101, the resulting plasmid was namedpME101-kivDll-yqhD-TT07.

The yqhD gene was PCR amplified from genomic DNA of the E. coli MG1655strain with the oligonucleotides yqhD F and yqhD R:

(SEQ ID NO 8) yqhD F T

cccagcaaagggagcaagtaatgaacaac

-   -   a region for addition of a SnaBI restriction site (italic bold        upper case)    -   a region (lower case) homologous to the E. coli MG1655 yqhD        region from 3153357 to 3153385

(SEQ ID NO 9) yqhD R a

cTTAGCGGGCGGCTTCGTATATAC

-   -   a region (upper case) homologous to the E. coli MG1655 yqhD        region from 3154540 to 3154518    -   a region for addition of a BglII restriction site (italic bold        lower case).

The PCR amplified fragment was cut with the restriction enzymes SnaBIand BglII and cloned into the SnaBI-BglII sites of the vectorpME101-kivDll-TT07 resulting in the vector pME101-kivDll-yqhD-TT07.

Example 2 Construction of Strains with Increased Ethylene Glycol PathwayFlux: MG1655 ΔsdaA ΔsdaB ΔpykF Ptrc18-gpmA Ptrc18-gpmB(pME101-kivDll-yqhD-TT07)

2.1 Construction of the MG1655 ΔsdaA ΔsdaB Strain

To delete the sdaA gene, the homologous recombination strategy describedby Datsenko & Wanner (2000) was used. This strategy allowed theinsertion of a chloramphenicol or a kanamycin resistance cassette, whiledeleting most of the genes concerned. For this purpose the followingoligonucleotides were used:

ΔsdaAF (SEQ ID NO 10)

gtcaggagtattatcgtgattagtctattcgacatgtttaaggtggggattggtccctcatcttcccataccgtagggccTGTAGGCTGGAGCTGCTTCG with

-   -   a region (lower case) homologous to the sequence        (1894941-1895020) of the sdaA gene (reference sequence on the        website http://genolist.pasteur.fr/Colibri/),    -   a region (upper bold case) for the amplification of the        kanamycin resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645),

ΔdaAR (SEQ ID NO 11)

GGGCGAGTAAGAAGTATTAGTCACACTGGACTTTGATTGCCAGACCACCGCGTGAGGTTTCGCGGTATTTGGCGTTCATGTCCCATATGAATATCCTCCTAAG with

-   -   a region (upper case) homologous to the sequence        (1896336-1896254) of the sdaA gene (reference sequence on the        website http://genolist.pasteur.fr/Colibri/),    -   a region (upper bold case) for the amplification of the        kanamycin resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645).

The oligonucleotides ΔsdaAF and ΔsdaAR were used to amplify thekanamycin resistance cassette from the pKD4 plasmid. The PCR productobtained was then introduced by electroporation into the strain MG 1655(pKD46). The kanamycin resistant transformants were selected and theinsertion of the resistance cassette was verified by PCR analysis withthe oligonucleotides sdaAF and sdaAR defined below. The strain retainedwas designated MG1655 ΔsdaA::Km. sdaAF (SEQ ID NO 12):cagcgttcgattcatctgcg (homologous to the sequence from 1894341 to1894360). sdaAR (SEQ ID NO 13):

GACCAATCAGCGGAAGCAAG (homologous to the sequence from 1896679 to1896660).

The kanamycin resistant transformants were then selected and theΔsdaA::Km was verified by PCR analysis with the previously definedoligonucleotides sdaAF and sdaAR. The strain retained was designatedMG1655 ΔsdaA::Km. Then the DsdaB::Cm was introduced into the strainMG1655 ΔsdaA::Km by transduction. The MG1655 ΔsdaB::Cm was firstconstructed using the same method as previously described with thefollowing oligonucleotides:

ΔsdaBF (SEQ ID NO 14)

cggcattggcccttccagttctcataccgttggaccaatgaaagcgggtaaacaatttaccgacgatctgattgcccgTGTAGGCTGGAGCTGCTTCG with

-   -   a region (lower case) homologous to the sequence        (2927627-2927705) of the sdaB gene (reference sequence on the        website http://genolist.pasteur.fr/Colibri/),    -   a region (upper bold case) for the amplification of the        chloramphenicol resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645),

ΔsdaBR (SEQ ID NO 15)

CGCAGGCAACGATCTTCATTGCCAGGCCGCCGCGAGAGGTTTCGCGGTACTTGGCGTTCATATCTTTACCTGTTTCGTACCATATGAATATCCTCCTTAG with

-   -   a region (upper case) homologous to the sequence        (2928960-2928881) of the sdaB gene (reference sequence on the        website http://genolist.pasteur.fr/Colibri/),    -   a region (upper bold case) for the amplification of the        chloramphenicol resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645). The        oligonucleotides ΔsdaBF and ΔsdaBR were used to amplify the        chloramphenicol resistance cassette from the plasmid pKD3. The        PCR product obtained was then introduced by electroporation into        the strain MG1655 (pKD46). The chloramphenicol resistant        transformants were then selected and the insertion of the        resistance cassette was verified by a PCR analysis with the        oligonucleotides sdaBF and sdaBR defined below. The strain        retained is designated MG1655 ΔsdaB::Cm. sdaBF (SEQ ID NO 16):        Gcgtaagtacagcggtcac (homologous to the sequence from 2927450 to        2927468). sdaBR (SEQ ID NO 17): CGATGCCGGAACAGGCTACGGC        (homologous to the sequence from 2929038 to 2929017). To        transfer the ΔsdaB::Cm, the method of phage P1 transduction was        used. The preparation of the phage lysate of the strain MG1655        ΔsdaB::Cm was used for the transduction into the strain MG1655        ΔsdaA::Km.

The chloramphenicol resistant transformants were then selected and theΔsdaB::Cm was verified by a PCR analysis with the previously definedoligonucleotides sdaBF and sdaBR. The strain retained was designatedMG1655 ΔsdaA::Km ΔsdaB::Cm. The kanamycin and chloramphenicol resistancecassettes were then be eliminated. The plasmid pCP20 carrying FLPrecombinase acting at the FRT sites of the kanamycin and thechloramphenicol resistance cassettes was then introduced into therecombinant sites by electroporation. After a series of cultures at 42°C., the loss of the kanamycin and chloramphenicol resistance cassetteswas verified by a PCR analysis with the same oligonucleotides as usedpreviously (sdaAF/sdaAR and sdaBF/sdaBR). The strain retained wasdesignated MG1655 DsdaA ΔsdaB.

2.2 Construction of Strain MG1655 ΔsdaA ΔsdaB ΔpykF

To delete the pykF gene, the homologous recombination strategy describedby Datsenko & Wanner (2000) was used. This strategy allowed theinsertion of a chloramphenicol or a kanamycin resistance cassette, whiledeleting most of the genes concerned. For this purpose the followingoligonucleotides were used:

ΔpykFF (SEQ ID NO 18)

cccatccttctcaacttaaagactaagactgtcatgaaaaagaccaaaattgtttgcaccatcggaccgaaaaccgaaTGTAGGCTGGAGCTGCTTCG

with

-   -   a region (lower case) homologous to the sequence        (1753689-1753766) of the pykF region (reference sequence on the        EcoGene website),    -   a region (upper case) for the amplification of the kanamycin        resistance cassette (reference sequence in Datsenko, K. A. &        Wanner, B. L., 2000, PNAS, 97: 6640-6645), ΔpykFR (SEQ ID NO 19)

ggacgtgaacagatgcggtgttagtagtgccgctcggtaccagtgcaccagaaaccataactacaacgtcacctttgtgCATATGAATATCCTCCTTAG

with

-   -   a region (upper case) homologous to the sequence        (1755129-1755051) of the pykF region (reference sequence on the        EcoGene website),    -   a region (upper case) for the amplification of the kanamycin        resistance cassette (reference sequence in Datsenko, K. A. &        Wanner, B. L., 2000, PNAS, 97: 6640-6645).

The oligonucleotides ΔpykFF and ΔpykFR were used to amplify thekanamycin resistance cassette from the plasmid pKD4. The PCR productobtained was then introduced by electroporation into the strain MG 1655(pKD46). The kanamycin resistant transformants were then selected andthe insertion of the resistance cassette was verified by a PCR analysiswith the oligonucleotides pykFF and pykFR defined below. The strainretained was designated MG1655 ΔpykF::Km. pykFF (SEQ ID NO 20):gcgtaaccttttccctggaacg (homologous to the sequence from 1753371 to1753392). pykFR (SEQ ID NO 21): gcgttgctggagcaacctgccagc (homologous tothe sequence from 1755518 to 1755495).

To transfer the ΔpykF::Km, the method of phage P1 transduction was used.The preparation of the phage lysate of the strain MG1655 ΔpykF::Km wasused for the transduction into the strain MG1655 ΔsdaA ΔsdaB.

The kanamycin resistant transformants were then selected and theΔpykF::Km was verified by a PCR analysis with the previously definedoligonucleotides pykFF and pykFR. The strain retained was designatedMG1655 ΔsdaA ΔsdaB ΔpykF::Km.

The kanamycin resistance cassette was then eliminated. The plasmid pCP20carrying FLP recombinase acting at the FRT sites of the kanamycinresistance cassette was then introduced into the recombinant sites byelectroporation. After a series of cultures at 42° C., the loss of thekanamycin resistance cassettes was verified by a PCR analysis with thesame oligonucleotides as used previously (sdaAF/sdaAR, sdaBF/sdaBR andpykFF/pykFR). The strain retained was designated MG1655 ΔsdaA ΔsdaBΔpykF.

2.3 Construction of Strain MG1655 ΔsdaA ΔsdaB ΔpykF Ptrc18-gpmAPtrc18-gpmB

To increase the level of 3-phosphoglycerate, the mutants Ptrc18-gpmA andPtrc18-gpmB are constructed. First, to reduce the expression of thephosphoglycerate mutase gpmA gene, the promoter is replaced by amodified constitutive trc promoter with weak activity.

The Ptrc18-gpmA is transferred into the MG1655 ΔsdaA ΔsdaB ΔpykF strainby transduction.

The strain MG1655 Ptrc18-gpmA::Km is first constructed using the samemethod as previously described with the following oligonucleotides:

Ptrc18-gpmAF (SEQ ID NO 22)

CCACTGACTTTCGCCATGACGAACCAGAACCAGCTTAGTTACAGCCATAATATACCTCCTTATTCCACACAgTATACGAGCCGGATGATTAATcGcCAACAGCTCTGTAGGCTGGAGCTGCTTCG with

-   -   a region (upper case) homologous to the sequence (786771-786819)        of the gpmA gene (reference sequence on the website        http://genolist.pasteur.fr/Colibri/),    -   a region (upper bold case) for the amplification of the        kanamycin resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645),    -   a region (upper italic case) for the trc promoter sequence where        the −35 and −10 boxes are underlined.

Ptrc18-gpmAR (SEQ ID NO 23)

ggttatgcgtaagcattgctgttgcttcgtcgcggcaatataatgagaattattatcattaaaagatgatttgaggagtaagtatCATATGAATATCCTCCTTAG with

-   -   a region (lower case) homologous to the sequence (786903-786819)        of the region upstream of the gpmA gene (reference sequence on        the website http://genolist.pasteur.fr/Colibri/),    -   a region (upper bold case) for the amplification of the        kanamycin resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645).

The oligonucleotides Ptrc18-gpmAF and Ptrc18-gpmAR are used to amplifythe kanamycin resistance cassette from the plasmid pKD4. The obtainedPCR product is then introduced by electroporation into the strain MG1655(pKD46), in which the expressed Red recombinase enzyme permits thehomologous recombination. The kanamycin resistant transformants are thenselected, and the insertion of the resistance cassette is verified by aPCR analysis with the oligonucleotides gpmAF and gpmAR defined below.The strain retained is designated MG1655 Ptrc18-gpmA::Km. gpmAF (SEQ IDNO 24): CCTTCCTCTTTCAGCAGCTTACC (homologous to the sequence from 786673to 786695). gpmAR (SEQ ID NO 25): cgacgatcagcgcaaagtgaaagg (homologousto the sequence from 787356 to 787333).

To transfer the modification Ptrc18-gpmA::Km, phage P1 transduction isused. The protocol followed is implemented in two steps, with first thepreparation of the phage lysate of the strain MG1655 Ptrc18-gpmA::Km,and second the transduction into the strain MG1655 ΔsdaA ΔsdaB ΔpykF.The construction of the strain is described above.

1—Preparation of the P1 Phage Lysate

-   -   Inoculation with 100 μl of an overnight culture of the strain        MG1655 Ptrc18-gpmA::Km of 10 ml of LB+Km 50 μg/ml+glucose        0.2%+CaCl2 5 mM. Incubation for 30 min at 37° C. with shaking.        —Addition of 100 μl of phage lysate P1 prepared on the strain        MG1655 (about 1.10⁹ phage/ml).    -   Shaking at 37° C. for 3 hours until all the cells were lysed.        Addition of 200 μl chloroform and vortexing.    -   Centrifugation for 10 min at 4500 g to eliminate cell debris.    -   Transfer of supernatant to a sterile tube and addition of 200 μl        chloroform.    -   Storage of lysate at 4° C.        2—Transduction    -   Centrifugation for 10 min at 1500 g of 5 ml of an overnight        culture of the MG1655 ΔsdaA ΔsdaB ΔpykF strain in LB medium.    -   Suspension of the cell pellet in 2.5 ml of 10 mM MgSO₄, 5 mM        CaCl2 Control tubes: 100 μl cells    -   100 μl phages P1 of strain MG1655 Ptrc18-gpmA::Km—Test tube: 100        μl of cells+100 μl of phages P1 of the strain MG1655        Ptrc18-gpmA::Km.    -   Incubation for 30 min at 30° C. without shaking. Addition of 100        μl of 1 M sodium citrate in each tube and vortex.    -   Addition of 1 ml of LB. —Incubation for 1 hour at 37° C. with        shaking.    -   Spreading on dishes LB+Km 50 mg/ml after centrifugation of tubes        for 3 min at 7000 rpm.    -   Incubation at 37° C. overnight.        3—Verification of the Strain

The kanamycin resistant transformants are then selected and themodification of the promoter Ptrc18-gpmA::Km is verified by a PCRanalysis with the oligonucleotides gpmAF and gpmAR previously described.The strain retained is designated MG1655 ΔsdaA ΔsdaB ΔpykFPtrc18-gpmA::Km. Then the Ptrc18-gpmB is transferred into the MG1655ΔsdaA ΔsdaB ΔpykF Ptrc18-gpmA::Km strain by transduction. The MG1655Ptrc18-gpmB::Cm is first constructed using the same method as previouslydescribed with the following oligonucleotides:

Ptrc18-gpmBR (SEQ ID NO 26)

CGGCGTTCC ACTGCGTTTCACCGTGGCGGACTAGGTATACCTGTAACATAATATACCTCCTTATTCCACACAgTATACGAGCCGGATGATTAATcGcCAACAGCTCTGTAGGCTGGA GCTGCTTCG with

-   -   a region (upper case) homologous to the sequence        (4631414-4631366) of the gpmB gene (reference sequence on the        website http://genolist.pasteur.fr/Colibri/),    -   a region (upper bold case) for the amplification of the        chloramphenicol resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97:6640-6645),    -   a region (upper italic case) for the trc promoter sequence where        the −35 and −10 boxes are underlined.

Ptrc18-gpmBF (SEQ ID NO 27)

agggattggtggtcgcacagacaacttggtgcataatcagcattactcagaaaattaacgttacagcagtatacggaaaaaaagcCATATGAATATCCTCCTTAG with

-   -   a region (lower case) homologous to the sequence        (4631280-4631365) of the region upstream of the gpmB gene        (reference sequence on the website        http://genolist.pasteur.fr/Colibri/),    -   a region (upper bold case) for the amplification of the        chloramphenicol resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645).

The oligonucleotides Ptrc18-gpmBF and Ptrc18-gpmBR are used to amplifythe chloramphenicol resistance cassette from the plasmid pKD3. The PCRproduct obtained is then introduced by electroporation into the strainMG1655 (pKD46), in which the Red recombinase enzyme expressed, permitsthe homologous recombination. The chloramphenicol resistanttransformants are then selected and the insertion of the resistancecassette is verified by a PCR analysis with the oligonucleotides gpmBFand gpmBR defined below. The strain retained is designated MG1655Ptrc18-gpmB::Cm gpmBF (SEQ ID NO 28): ccttacgaccaatctcatcaataccgg(homologous to the sequence from 4630906 to 4630932). gpmBR (SEQ ID NO29): GCAATACCATGACTCACCAGC (homologous to the sequence from 4631823 to4631803).

To transfer the modification Ptrc18-gpmB::Cm, phage P1 transduction isused. Phage lysate of the strain MG1655 Ptrc18-gpmB::Cm is used for thetransduction into the strain MG1655 ΔsdaA ΔsdaB ΔpykF Ptrc18-gpmA::Km.The chloramphenicol resistant transformants are then selected and thePtrc18-gpmB::Cm is verified by a PCR analysis with the previouslydefined oligonucleotides gpmBF and gpmBR. The strain retained isdesignated MG1655 ΔsdaA ΔsdaB ΔpykF Ptrc18-gpmA::Km Ptrc18-gpmB::Cm. Thekanamycin and chloramphenicol resistance cassettes can then beeliminated. The plasmid pCP20 carrying FLP recombinase acting at the FRTsites of the kanamycin and the chloramphenicol resistance cassettes isthen introduced into the recombinant sites by electroporation. After aseries of cultures at 42° C., the loss of the kanamycin andchloramphenicol resistance cassettes is verified by a PCR analysis withthe same oligonucleotides as used previously (gpmAF/gpmAR andgpmBF/gpmBR). The strain retained is designated MG1655 ΔsdaA ΔsdaB ΔpykFPtrc18-gpmA Ptrc18-gpmB.

2.4 Construction of the MG1655 ΔsdaA ΔsdaB ΔpykF Ptrc18-gpmA Ptrc18-gpmB(pME101-kivDll-yqhD-TT07) Strain

The pME101-kivDll-yqhD-TT07 plasmid is then introduced into the strainMG1655 ΔsdaA ΔsdaB ΔpykF Ptrc18-gpmA Ptrc18-gpmB.

Example 3 Construction of Strains with Increased 1,3-Propanediol PathwayFlux: MG1655 ΔmetA ΔpykF ΔthrLABC::TT07-Ptrc-thrA*1(pME101-kivDll-yqhD-TT07) (pMA-Aaoro)

3.1 Construction of Strain MG1655 ΔmetA

To delete the metA gene, the homologous recombination strategy describedby Datsenko & Wanner (2000) was used. This strategy allows the insertionof a chloramphenicol or a kanamycin resistance cassette, while deletingmost of the genes concerned. For this purpose the followingoligonucleotides were used:

ΔmetAF (SEQ ID NO 30):

ttcgtgtgccggacgagctacccgccgtcaatttatgcgtgaagaaaacgtattgtgatgacaacttacgtgcgtaTGTAGGCTGGAGCTGCTTCG

with

-   -   a region (lower case) homologous to the sequence        (4212310-4212389) of the metA region (reference sequence on the        EcoGene website),    -   a region (upper case) for the amplification of the kanamycin        resistance cassette (reference sequence in Datsenko, K. A. &        Wanner, B. L., 2000, PNAS, 97: 6640-6645),

ΔmetAR (SEQ ID NO 31):

atccagcgttggattcatgtgccgtagatcgtatggcgtgatctggtagacgtaatagttgagccagttggtaaacagtaCATATGAATATCCTCCTTAG

with

-   -   a region (upper case) homologous to the sequence        (4213229-4213150) of the metA region sequence on the EcoGene        website),    -   a region (upper case) for the amplification of the kanamycin        resistance cassette (reference sequence in Datsenko, K. A. &        Wanner, B. L., 2000, PNAS, 97: 6640-6645).

The oligonucleotides ΔmetAF and ΔmetAR were used to amplify thekanamycin resistance cassette from the plasmid pKD4. The PCR productobtained was then introduced by electroporation into the strain MG 1655(pKD46). The kanamycin resistant transformants were then selected andthe insertion of the resistance cassette was verified by a PCR analysiswith the oligonucleotides metAF and metAR defined below. The strainretained was designated MG1655 ΔmetA::Km. metAF (SEQ ID NO 32):tcaccttcaacatgcaggctcgacattggc (homologous to the sequence from 4212203to 4212232). metAR (SEQ ID NO 33): ataaaaaaggcacccgaaggtgcctgaggt(homologous to the sequence from 4213301 to 4213272).

The kanamycin resistance cassette was then eliminated. The plasmid pCP20carrying FLP recombinase acting at the FRT sites of the kanamycinresistance cassette was then introduced into the recombinant sites byelectroporation. After a series of cultures at 42° C., the loss of thekanamycin resistance cassette was verified by a PCR analysis with thesame oligonucleotides as used previously (metAF/metAR). The strainretained was designated MG1655 ΔmetA.

3.2 Construction of Strain MG1655 ΔmetA ΔpykF

To transfer the ΔpykF::Km, phage P1 transduction was used. Thepreparation of the phage lysate of the strain MG1655 ΔpykF::Km(described above) was used for the transduction into the strain MG1655ΔmetA.

Kanamycin resistant transformants are then selected and the ΔpykF::Kmwas verified by a PCR analysis with the previously definedoligonucleotides pykFF and pykFR. The strain retained was designatedMG1655 ΔmetA DpykF::Km.

3.3 Construction of Strain MG1655 DmetA DpykF DthrLABC::TT07-Ptrc-thrA*1

To increase the expression of the feedback resistant allele of theaspartokinase/homoserine dehydrogenase, thrA*1, the following plasmidswere constructed: pSB1 to obtain the thrA*1 and pSB2 to replace thethrLABC operon by the Ptrc-thrA*1 allele.

The plasmid pSB1 is derived from plasmid pCL1920 (Lerner & Inouye, 1990,NAR 18, 15 p 4631) and harbors the aspartokinase/homoserine thrA*allelewith reduced feed-back resistance to threonine (Lee et al. 2003 J.Bacteriol. 185, 18 pp. 5442-5451) expressed from the promoter Ptrc. Forthe construction of pSB1, thrA was PCR amplified from genomic DNA usingthe following oligonucleotides:

BspHIthrA (SEQ ID NO 34):

TTATCATGAgagtgttgaagttcggcggtacatcagtggc

with

-   -   a region (lower case) homologous to the sequence (341-371) of        the thrA gene (reference sequence on the website        http://www.ecogene.org/),    -   a region (upper case) for BspHI restriction site and        extra-bases,

SmaIthrA (SEQ ID NO 35):

TTACCCGGGccgccgccccgagcacatcaaacccgacgc

with

-   -   a region (lower case) homologous to the sequence (2871-2841) of        the thrA gene (reference sequence on the EcoGene website),    -   a region (upper case) for SmaI restriction site and extra-bases.

The PCR amplified fragment was cut with the restriction enzymes BspHIand SmaI and cloned into the NcoI/SmaI sites of the vector pTRC99A(Stratagene). For the expression from a low copy vector the pME101plasmid was constructed as follows. The pCL1920 plasmid was PCRamplified using the oligonucleotides PME101F and PME101R and theBstZ17I-XmnI fragment from the pTRC99A vector harboring the lad gene andthe Ptrc promoter was inserted into the amplified vector. The resultingvector and the vector harboring the thrA gene were restricted by ApaIand SmaI and the thrA containing fragment was cloned into the vectorpME101. To relieve ThrA from feedback inhibition the mutation thrAS345Fwas introduced by site-directed mutagenesis (Stratagene) using theoligonucleotides ThrA SF for and ThrA SF rev, resulting in the vectorpSB1.

PME101F (SEQ ID NO 36) ccgacagtaagacgggtaagcctg PME101R (SEQ ID NO 37)agcttagtaaagccctcgctag ThrA SF for (SEQ ID NO 38)CGTATTTCCGTGGTGCTGATTACGCAATTCTCTTCCGAGTACTCAATC AGTTTCTGC ThrA SF rev(SEQ ID NO 39) GCAGAAACTGATTGAGTACTCGGAAGAGAATTGCGTAATCAGCACCACGGAAATACG

To delete the thrLABC operon and replace it by Ptrc-thrA*1 allele, thehomologous recombination strategy described by Datsenko & Wanner (2000)was used. This strategy allows the insertion of a chloramphenicol or akanamycin resistance cassette but also of additional DNA, while deletingmost of the genes concerned. For this purpose, the following plasmid wasconstructed, pSB2.

The plasmid pSB2 is derived from plasmid pUC18 (Norrander et al., Gene26 (1983), 101-106) and harbours the chloramphenicol resistance cassetteassociated to Ptrc-thrA*1 allele, both cloned between the upstreamregion of thrL and the downstream region of thrC.

For the construction of pSB2, the upstream region of thrL and thedownstream region of thrC were PCR amplified from genomic DNA using thefollowing oligonucleotides:

HpaIupthrLF (SEQ ID NO 40)

CGTAGTTAACGAATTCccaactagttgcatcatacaactaataaacgtgg

with

-   -   a region (lower case) homologous to the sequence        (4638698-4638731) of the thrL region (reference sequence on the        EcoGene website),    -   a region (upper case) for HpaI and EcoRI restriction site and        extra-bases. BstZ17IupthrLR (SEQ ID NO 41)

CCCGGGGGAGGCGCCCGCGGATCCCGGTATACCAGAAAGGCCCACCCGAAGGTGAGCCAGGAaggtaaccagttcagaagagetatcag

with

-   -   a region (lower case) homologous to the sequence (87-60) of the        thrL region (reference sequence on the EcoGene website),    -   a region (upper bold case) for T7te transcriptional terminator        sequence (Harrington K. J., Laughlin R. B. and Liang S. Proc        Natl Acad Sci USA. 2001 Apr. 24; 98(9):5019-24),    -   a region (upper case) for the multiple cloning site composed of        BstZ17I, BamHI, SfoI and SmaI restriction sites.

BamHIdownthrCF (SEQ ID NO 42)

TCCTGGCTCACCTTCGGGTGGGCCTTTCTGGTATACCGGGATCCGCGGGCGCCTCCCCCGGGaatctattcattatctcaatcaggccggg

with

-   -   a region (lower case) homologous to the sequence (5021-5049) of        the thrC region (reference sequence on the EcoGene website),    -   a region (upper bold case) for T7te transcriptional terminator        sequence (Harrington K. J., Laughlin R. B. and Liang S. Proc        Natl Acad Sci USA. 2001 Apr. 24; 98(9):5019-24),    -   a region (upper case) for the multiple cloning site composed of        BstZ17I, BamHI, SfoI and

SmaI restriction sites.

HpaIdownthrCR (SEQ ID NO 43)

CGTAGTTAACGAATTCgagaatgcccgagggaaagatctg

with

-   -   a region (lower case) homologous to the sequence (6054-6031) of        the thrC region (reference sequence on the EcoGene website),    -   a region (upper case) for HpaI and EcoRI restriction site and        extra-bases. First, the “upthrL” and “downthrC” fragments were        PCR amplified from MG1655 genomic DNA using        HpaIupthrLF/BstZ17IupthrLR and BamHIdownthrCF/HpaIdownthrCR        oligonucleotides, respectively. Secondly, the“upthrL-downthrC”        fragment was amplified from “upthrL” and “downthrC” PCR        fragments (that possess an overlapping region composed of a T7Te        transcriptional terminator and the multiple cloning site        composed of BstZ17I, BamHI, SfoI and SmaI restriction sites)        using HpaIupthrLF/HpaIdownthrCR oligonucleotides. The        “upthrL-downthrC” PCR fragment was cut with the restriction        enzyme HpaI and cloned into the EcoRI/SfoI sites of the pUC18        vector, giving the pUC18-DthrLABC::TT07-SMC plasmid.

Then, the chloramphenicol resistance cassette was PCR amplified frompKD3 vector using the following oligonucleotides:

BstZ17ICmF (SEQ ID NO 44)

GGGGTATACtgtaggctggagctgcttcg

with

-   -   a region (lower case) for the amplification of the        chloramphenicol resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645),    -   a region (upper case) for BstZ17I restriction site and        extra-bases.

BamHICmR (SEQ ID NO 45)

CGCGGATCCcatatgaatatcctccttag

with

-   -   a region (lower case) for the amplification of the        chloramphenicol resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645),    -   a region (upper case) for BamHI restriction site and        extra-bases.

The PCR fragment was cut with the restriction enzymes BstZ17I and BamHIand cloned into the BstZ17I/BamHI sites of the pUC18-ΔthrLABC::TT07-SMCplasmid, giving the pUC18-ΔthrLABC::TT07-SMC::Cm plasmid.

Finally, the Ptrc-thrA*1 allele was cut from the pSB1 plasmid with therestriction enzymes SfoI and SmaI and cloned into the SfoI/SmaI sites ofthe pUC18-ΔthrLABC::TT07-SMC::Cm plasmid, giving thepUC18-ΔthrLABC::TT07-Ptrc-thrA*1::Cm plasmid or pSB2.

The ΔthrLABC::TT07-Ptrc-thrA*1::Cm fragment was obtained by cutting thepSB2 plasmid with EcoRI restriction enzyme and was then introduced byelectroporation into the strain MG1655 (pKD46), in which the expressedRed recombinase enzyme permits the homologous recombination. Thechloramphenicol resistant transformants are then selected, and theinsertion of the resistance cassette is verified by a PCR analysis withthe oligonucleotides thrA*1F and thrA*1R defined below. The strainretained is designated MG1655 ΔthrLABC::TT07-Ptrc-thrA*1::Cm. thrA*1F(SEQ ID NO 46): cgtgttgcgtgttaccaactcg (homologous to the sequence(4638276-4638297) of the thrL region (reference sequence on the EcoGenewebsite)). thrA*1R (SEQ ID NO 47) cggaaactgacgcctccgcag (homologous tothe sequence (6345-6325) of the thrC region (reference sequence on theEcoGene website)).

Recombinant plasmids were verified by DNA sequencing.

To transfer the ΔthrLABC::TT07-Ptrc-thrA*1::Cm, phage P1 transduction isused. The preparation of the phage lysate of the strain MG1655ΔthrLABC::TT07-Ptrc-thrA*1::Cm is used for the transduction into thestrain MG1655 ΔmetA DpykF::Km.

The kanamycin and chloramphenicol resistant transformants are thenselected and the ΔthrLABC::TT07-Ptrc-thrA*1::Cm is verified by a PCRanalysis with the previously defined oligonucleotides thrA*1F andthrA*1R. The strain retained is designated MG1655 ΔmetA ΔpykF::KmΔthrLABC::TT07-Ptrc-thrA*1::Cm.

The kanamycin and chloramphenicol resistance cassettes can then beeliminated. The plasmid pCP20 carrying FLP recombinase acting at the FRTsites of the kanamycin and chloramphenicol resistance cassettes is thenintroduced into the recombinant sites by electroporation. After a seriesof cultures at 42° C., the loss of the kanamycin and chloramphenicolresistance cassettes is verified by a PCR analysis with the sameoligonucleotides as used previously (metAF/metAR, pykF/pykFR andthrA*1F/thrA*1R). The strain retained is designated MG1655 ΔmetA ΔpykFΔthrLABC::TT07-Ptrc-thrA*1.

3.4 Construction of Plasmid pMA-aaoro

A synthetic gene of the Rhodococcus opacus aao gene coding for aminoacid oxidase was prepared by the Geneart (Germany). The codon usage andGC content of the genes was adapted to Escherichia coli according to thesupplier matrix. Expression of the synthetic gene was driven by aconstitutive Ptrc promoter. The construct was cloned into supplier's pMAvector and verified by sequencing.

Ptrc01-aaoro:

restriction sites (KpnI, EcoRI, SmaI) (SEQ ID NO 48): ggtaccgaattccccgggPtrc01 promoter and RBS (SEQ ID NO 49):gagctgttgacaattaatcatccggctcgtataatgtgtggaaggatc ccccgggtaaggaggttataaaaoro gene sequence optimized for E. coli (AY053450.) (SEQ ID NO 50):atggcatttacccgtcgcagctttatgaaaggtctgggtgcaaccggtggtgcaggtctggcatatggtgcaatgagcaccctgggtctggcaccgtctacagcagcaccggcacgtacctttcagccgctggcagccggtgatctgattggtaaagtgaaaggtagccatagcgttgttgttctgggtggtggtccggcaggtctgtgtagcgcatttgaactgcagaaagccggttataaagttaccgttctggaagcacgtacccgtccgggtggtcgtgtttggaccgcacgtggtggtagcgaagaaaccgatctgagcggtgaaacccagaaatgtacctttagcgaaggccatttttataatgttggtgccacccgtattccgcagagccatattaccctggattattgtcgcgaactgggtgttgaaattcagggtttcggcaatcaaaatgccaatacctttgtgaattatcagagcgataccagcctgagcggtcagagcgttacctatcgtgcagcaaaagcagatacctttggctatatgagcgaactgctgaaaaaagcaaccgatcagggtgcactggatcaggttctgagccgtgaagataaagatgcactgagcgaatttctgagcgattttggtgatctgtctgatgatggtcgttatctgggtagcagccgtcgtggttatgatagcgaaccgggtgccggtctgaattttggcaccgaaaaaaaaccgtttgccatgcaggaagttattcgtagcggtattggtcgcaattttagctttgattttggctatgatcaggccatgatgatgtttacaccggttggtggtatggatcgtatttattatgcctttcaggatcgtattggcactgataacatcgtgttcggtgccgaagttaccagcatgaaaaatgttagcgaaggtgtgaccgttgaatataccgcaggcggtagcaaaaaaagcattaccgcagattatgccatttgtaccattcctccgcatctggttggtcgtctgcagaataatctgcctggtgatgttctgaccgcactgaaagcagcaaaaccgagcagcagcggtaaactgggtattgaatatagccgtcgttggtgggaaaccgaagatcgcatttatggtggtgcaagcaataccgataaagatattagccagattatgtttccgtatgatcattataatagcgatcgtggtgttgttgttgcatattatagctctggtaaacgccaggaagcatttgaaagcctgacccatcgtcagcgtctggcaaaagcaattgcagaaggcagcgaaattcacggcgaaaaatatacccgtgatattagcagcagctttagcggtagctggcgtcgtaccaaatatagcgaaagcgcatgggcaaattgggcaggtagcggtggttctcatggtggtgcagccactccggaatatgaaaaactgctggaaccggtggataaaatttattttgccggtgatcatctgagcaatgcaatcgcatggcagcatggtgcactgaccagcgcacgtgatgttgttacccatattcatgaa cgtgttgcacaggaagcctaarestriction sites (BglII, EcoRV, PacI, SacI,XbaI, HindIII) (SEQ ID NO 51): gatctgatatcttaattaagagctctctagaaagctt

3.5 Construction of Strain MG1655 ΔmetA ΔpykF ΔthrLABC::TT07-Ptrc-thrA*1(pME101-kivDll-yqhD-TT07) (pMA-aaoro)

The pME101-kivDll-yqhD-TT07 and the pMA-aaoro plasmids are thenintroduced into the strain MG1655 ΔmetA ΔpykFΔthrLABC::TT07-Ptrc-thrA*1.

Example 4 Construction of Strains with Increased 1,4-Butanediol PathwayFlux: MG1655 ΔsucCD ΔaceBAK ΔarcA ΔgdhA(pUC19-Ptrc01/OP01/RBS01-adhE2ca-prpE-TT02) (pME101-kivDll-yqhD-TT07)

4.1 Construction of Strain MG1655 ΔaceBAK ΔsucCD

To delete the aceBAK genes, the homologous recombination strategydescribed by Datsenko & Wanner (2000) was used. This strategy allows theinsertion of a chloramphenicol or a kanamycin resistance cassette, whiledeleting most of the genes concerned. For this purpose the followingoligonucleotides were used:

ΔaceBAKF (SEQ ID NO 52):

ctggattcacaaggccgtatggcgagcaggagaagcaaattettactgccgaageggtagaatttctgactgagaggtTGTAGGCTGGAGCTGCTTCG

with

-   -   a region (lower case) homologous to the sequence        (4213531-4213610) of the aceB region (reference sequence on the        EcoGene website),    -   a region (upper bold case) for the amplification of the        kanamycin resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645),

ΔaceBAKR (SEQ ID NO 53):

aacatcttccacatgcccttcacgtatgcggttttgtagtgcgcgccagtaatcagcgcggaacaggtcggcgtgcatcCATATGAATATCCTCCTTAG

with

-   -   a region (upper case) homologous to the sequence        (4218298-4218220) of the aceK region (reference sequence on the        EcoGene website),    -   a region (upper bold case) for the amplification of the        kanamycin resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645).

The oligonucleotides ΔaceBAKF and ΔaceBAKR are used to amplify thekanamycin resistance cassette from the plasmid pKD4. The PCR productobtained is then introduced by electroporation into the strain MG 1655(pKD46). The kanamycin resistant transformants are then selected and theinsertion of the resistance cassette is verified by a PCR analysis withthe oligonucleotides aceBAKF and aceBAKR defined below. The strainretained is designated MG1655 ΔmetA::Km. aceBAKF (SEQ ID NO 54):cgttaagcgattcagcaccttacc (homologous to the sequence from 4213251 to4213274). aceBAKR (SEQ ID NO 55): aacgcattacccactctgtttaatacg(homologous to the sequence from 4218728 to 4218702).

To delete the sucCD genes, the homologous recombination strategydescribed by Datsenko & Wanner (2000) was used. This strategy allows theinsertion of a chloramphenicol or a kanamycin resistance cassette, whiledeleting most of the genes concerned. For this purpose the followingoligonucleotides were used:

ΔsucCDF (SEQ ID NO 56):

tttttgcccgctatggcttaccagcaccggtgggttatgcctgtactactccgcgcgaagcagaagaagccgcttcaaaaCATATGAATATCCTCCTTAG

with

-   -   a region (lower case) homologous to the sequence (762268-762347)        of the sucC region (reference sequence on the EcoGene website),    -   a region (upper bold case) for the amplification of the        chloramphenicol resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645),

ΔsucCDR (SEQ ID NO 57):

atatccgccaggctgcgaacggttttcacgcctgcggcttccagagcagcgaatttctcatccgcagtccctttaccggcTGTAGGCTGGAGCTGCTTCG

with

-   -   a region (upper case) homologous to the sequence (764241-764168)        of the sucD region (reference sequence on the EcoGene website),    -   a region (upper bold case) for the amplification of the        chloramphenicol resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645).

The oligonucleotides DsucCDF and DsucCDR are used to amplify thechloramphenicol resistance cassette from the plasmid pKD3. The PCRproduct obtained is then introduced by electroporation into the strainMG 1655 (pKD46). The chloramphenicol resistant transformants are thenselected and the insertion of the resistance cassette is verified by aPCR analysis with the oligonucleotides sucCDF and sucCDR defined below.The strain retained is designated MG1655 ΔsucCD::Cm. sucCDF (SEQ ID NO58): tcgcgaatccgtgggcttcctggtaacg (homologous to the sequence from761887 to 761914). sucCDR (SEQ ID NO 59: cctctgatgccaaccgaagagatgagccg(homologous to the sequence from 764555 to 764527).

To transfer the ΔaceBAK::Km, the method of phage P1 transduction isused. The preparation of the phage lysate of the strain MG1655DaceBAK::Km is used for the transduction into the strain MG1655ΔsucCD::Cm.

The kanamycin and chloramphenicol resistant transformants are thenselected and the ΔaceBAK::Km is verified by a PCR analysis with thepreviously defined oligonucleotides aceBAKF and aceBAKR. The strainretained is designated MG1655 ΔsucCD::Cm ΔaceBAK::Km.

The kanamycin and chloramphenicol resistance cassettes can then beeliminated. The plasmid pCP20 carrying FLP recombinase acting at the FRTsites of the kanamycin and chloramphenicol resistance cassettes is thenintroduced into the recombinant sites by electroporation. After a seriesof cultures at 42° C., the loss of the kanamycin and chloramphenicolresistance cassettes is verified by a PCR analysis with the sameoligonucleotides as used previously (aceBAKF/aceBAKR, andsucCDF/sucCDR). The strain retained is designated MG1655 ΔsucCD ΔaceBAK.

4.2 Construction of Strain MG1655 ΔsucCD ΔaceBAK ΔarcA ΔgdhA

To delete the arcA gene, the homologous recombination strategy describedby Datsenko & Wanner (2000) was used. This strategy allows the insertionof a chloramphenicol or a kanamycin resistance cassette, while deletingmost of the genes concerned. For this purpose the followingoligonucleotides were used:

ΔarcAF (SEQ ID NO 60):cccgcacattatatcgttgaagacgagttggtaacacgcaacacgttgaaaagtattttcgaageggaaggctatgTGTAGGCTGGAGCTGCTTCG

with

-   -   a region (lower case) homologous to the sequence        (4638322-4638245) of the arcA region (reference sequence on the        EcoGene website),    -   a region (upper bold case) for the amplification of the        kanamycin resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645),

ΔarcAR (SEQ ID NO 61):

ccagatcaccgcagaagcgataaccttcaccgtgaatggtggcgatgatttccggcgtatccggcgtagattcgaaatgCATATGAATATCCTCCTTAG

with

-   -   a region (upper case) homologous to the sequence        (4637621-4637699) of the arcA region (reference sequence on the        EcoGene website),    -   a region (upper bold case) for the amplification of the        kanamycin resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645).

The oligonucleotides DarcAF and DarcAR are used to amplify the kanamycinresistance cassette from the plasmid pKD4. The PCR product obtained isthen introduced by electroporation into the strain MG 1655 (pKD46). Thekanamycin resistant transformants are then selected and the insertion ofthe resistance cassette is verified by a PCR analysis with theoligonucleotides arcAF and arcAR defined below. The strain retained isdesignated MG1655 ΔarcA::Km. arcAF (SEQ ID NO 62): cgacaattggattcaccacg(homologous to the sequence from 4638746 to 4638727). arcAR (SEQ ID NO63): gcggtattgaaaggttggtgc (homologous to the sequence from 4637308 to4637328).

To transfer the ΔarcA::Km, phage P1 transduction is used. Phage lysateof the strain MG1655 DarcA::Km is used for the transduction into thestrain MG1655 ΔsucCD ΔaceBAK.

The kanamycin resistant transformants are then selected and theΔarcA::Km is verified by a PCR analysis with the previously definedoligonucleotides arcAF and arcAR. The strain retained is designatedMG1655 ΔsucCD ΔaceBAK ΔarcA::Km.

To delete the gdhA gene, the homologous recombination strategy describedby Datsenko & Wanner (2000) was used. The oligonucleotides ΔgdhAF andΔgdhAR are used to amplify the chloramphenicol resistance cassette fromthe plasmid pKD3.

ΔgdhAF (SEQ ID NO 64):taaacaacataagcacaatcgtattaatatataagggttttatatctatgTGTAGGCTGGAGCTGCTTCGwith

-   -   a region (lower case) homologous to the sequence (1840348        to 1840397) upstream of the gene gdhA        (http://ecogene.org/blast.php)    -   a region (upper bold case) for the amplification of the        chloramphenicol resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645),

ΔgdhAR (SEQ ID NO 65):taagcgtagcgccatcaggcatttacaacttaaatcacaccctgcgccagCATATGAATATCCTCCTTAG

with:

-   -   a region (lower case) homologous to the sequence (1841767        to 1841718) to the end and the downstream region of the gdhA        gene (http://ecogene.org/blast.php)    -   a region (upper bold case) for the amplification of the        chloramphenicol resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645),

The PCR product obtained is then introduced by electroporation into theMG1655 (pKD46) strain. The chloramphenicol resistant transformants arethen selected and the insertion of the resistance cassette is verifiedby a PCR analysis with oligonucleotides Ptrc-gdhAverF and gdhA R.

Ptrc-gdhAverF(SEQ ID NO 66): CCTTAACGTTATTGTCTCTGC

-   -   a region homologous to the sequence (1840168-1840188) of the        region upstream of the gdhA gene (http://ecogene.org/blast.php)

gdhA R(SEQ ID NO 67): GGAGGAAGCCCCAGAGCAGG

-   -   a region homologous to the sequence (1842274-1842293) of the        region downstream of the gdhA gene        (http://ecogene.org/blast.php).

The strain obtained was named MG1655 ΔgdhA::Cm.

To transfer the ΔgdhA::Cm, phage P1 transduction is used. Phage lysateof the strain MG1655 ΔgdhA::Cm is used for the transduction into thestrain MG1655 ΔsucCD

ΔaceBAK ΔarcA::Km.

The kanamycin and chloramphenicol resistant transformants are thenselected and the ΔgdhA::Cm is verified by a PCR analysis with thepreviously defined oligonucleotides Ptrc-gdhAverF and gdhA R. The strainretained is designated MG1655 ΔsucCD ΔaceBAK ΔarcA::Km ΔgdhA:: Cm.

The kanamycin and chloramphenicol resistance cassettes can then beeliminated. The plasmid pCP20 carrying FLP recombinase acting at the FRTsites of the kanamycin and chloramphenicol resistance cassettes is thenintroduced into the recombinant sites by electroporation. After a seriesof cultures at 42° C., the loss of the kanamycin and chloramphenicolresistance cassettes is verified by a PCR analysis with the sameoligonucleotides as used previously (aceBAKF/aceBAKR, sucCDF/sucCDR,arcAF/arcAR, and gdhAverF/gdhA R). The strain retained is designatedMG1655 ΔsucCD ΔaceBAK ΔarcA ΔgdhA.

4.3 Construction of a Plasmid for Overexpression of the BifunctionalAcetaldehyde-CoA/alcohol dehydrogenase adhE2 of Clostridiumacetobutylicum and the propionyl-CoA synthetase prpE gene of Escherichiacoli: pUC19-Ptrc01/OP01/RBS01-adhE2ca-prpE-TT02

The adhE2 gene from Clostridium acetobutylicum coding for thebifunctional acetaldehyde-CoA/alcohol dehydrogenase was cloned in theplasmid pUC19. The prpE gene coding for the propionyl-CoA synthetase wascloned upstream adhE2.

The adhE2 gene was PCR amplified from the megaplasmid pSol1 of theClostridium acetobutylicum strain ATCC824 (position 33722 to 36298) withthe oligonucleotides adhE2Ca F and adhE2Ca R.

adhE2Ca F (SEQ ID NO 68):

ggtaccggatccgggcccgagagttgacaattaatcatccggctcgtataatgtgtggaattgtgagcggataacaattTACGTAtaaggaggtatattATGAAAGTTACAAATCAAAAAGAAC with

-   -   region (bold lower case) for the addition of KpnI, BamHI, ApaI        restriction site    -   region (underlined lower case) for the addition of the promoter        Ptrc01    -   region (italic lower case) for the addition of an operator        sequence OP01    -   region (bold upper case) for the addition of SnaBI restriction        site    -   region (lower case) for the addition RBS01 sequence    -   region (underlined upper case) homologous the C. acetobutylicum        adhE2 region from 33722 to 33752

adhE2Ca R (SEQ ID NO 69):

GAGCTCAAGCTTaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttgatgcctagggctagctctagattaaTTAAAATGATTTTATATAGATATCCTTAAGTTCAC, with

-   -   region (upper case) for the addition of the HindIII, SacI        restriction site.    -   region (underlined bold lower case) for the addition of the        terminator TT02    -   region (italic case) for the addition of the PacI, XbaI, NheI,        AvrII restriction site    -   region (underlined upper case) homologous the C. acetobutylicum        adhE2 region from 36264 to 36298).

This PCR fragment was digested with BamHI and HindIII and cloned intothe vector pUC19 digested with the same restriction enzymes. The plasmidobtained was named pUC19-Ptrc01/OP01/RBS01-adhE2ca-TT02

To amplify the prpE gene, a PCR are carried out using chromosomal DNA ofE. coli as template and the primers prpE F and prpE R.

prep F (SEQ ID NO 70): tctagaggatcc aagttcaacaggagagcattatg

-   -   a region (bold underlined case) for the addition of the        restriction sites XbaI and BamHI    -   a region homologous (lower case) to the region from 351910 to        351932 (http://ecogene.org/blast.php)

prep R_(SEQ ID NO 71): ggatccgctagccctaggtacgta ctactcttccatcgcctggc

-   -   a region (bold underlined case) for the addition of the        restriction sites BamHI, NheI, AvrII, SnaBII    -   a region (lower case) homologous to the region from 353816 to        353797 (http://ecogene.org/blast.php)

This PCR fragment was digested with XbaI and NheI and cloned into thevector pUC19-Ptrc01/OP01/RBS01-adhE2ca-TT02 digested with the samerestriction enzymes. The plasmid obtained was namedpUC19-Ptrc01/OP01/RBS01-adhE2ca-prpE-TT02.

4.4 Construction of the MG1655 ΔsucCD ΔaceBAK ΔarcA ΔgdhA(pUC19-Ptrc01/OP01/RBS01-adhE2ca-prpE-TT02) (pME101-kivDll-yqhD-TT07)Strain

The pUC19-Ptrc01/OP01/RBS01-adhE2ca-prpE-TT02 and thepME101-kivDll-yqhD-TT07 plasmids are then introduced into the strainMG1655 ΔsucCD ΔaceBAK ΔarcA ΔgdhA.

Example 5 Fermentation of Ethylene Glycol Producing Strains inErlenmeyer Flasks

Performances of strains were assessed in 500 ml baffled Erlenmeyer flaskcultures using modified M9 medium (Anderson, 1946, Proc. Natl. Acad.Sci. USA 32:120-128) that was supplemented with 10 g/l MOPS and 10 g/lglucose and adjusted at pH 6.8. Spectinomycin was added if necessary ata concentration of 50 mg/l and/or Chloramphenicol was added if necessaryat a concentration of 30 mg/l. A 24 h preculture was used to inoculate a50 ml culture to an OD600 nm of about 0.3. The cultures were kept on ashaker at 37° C. and 200 rpm until the glucose in the culture medium wasexhausted. At the end of the culture, glucose and major products wereanalyzed by HPLC using a Biorad HPX 97H column for the separation and arefractometer for the detection. Production of Ethylene Glycol wasconfirmed by gas chromatography/mass spectrometry (GC/MS) with a HewlettPackard 6890 Series gas chromatograph coupled to a Hewlett Packard 5973Series mass selective detector (EI) and a HP-INNOWax column (25 mlength, 0.20 mm i.d., 0.20 micron film thickness). The retention timeand mass spectrum of Ethylene Glycol generated were compared to that ofauthentic Ethylene Glycol.

Comparison of the performances between the production strain and areference strain is given in table below. (see below for theconstruction of the producing strain)

[ethylene glycol] Culture_ref Strain_genotype (mM) FbDI_0180 MG1655DpykF nd FbDI_0184 MG1655 DpykF (pME101-kivDll- 0.63 yqhD-yeaB-TT07)(pCC1BAC-serA) nd: not detected

Example 6 Construction of Strain with Increased Ethylene Glycol PathwayFlux: MG1655 ΔpykF (pME101-kivDll-yqhD-yeaB-TT07) (pCC1BAC-serA)

6.1 Construction of a Plasmid for the Overexpression of the HydroxyKeto-Acid Decarboxylase kivD of Lactococcus lactis, the Hydroxy AldehydeReductase yqhD and the Phosphohydroxy Pyruvate Phosphatase yeaB Genes ofEscherichia coli: pME101-kivDll-yqhD-yeaB-TT07 Plasmid

The yeaB containing fragment was restricted by XbaI and BglII and clonedinto the vector pME101-kivDll-yqhD restricted by the same restrictionenzymes, the resulting plasmid was named pME101-kivDll-yqhD-yeaB-TT07.

The yeaB gene was PCR amplified from genomic DNA of the E. coli MG1655strain with the oligonucleotides yeaB F and yeaB R:

yeaB F (SEQ ID NO 72) AGCT

TA

AT

taaggaggtatattATGGAA TACCGTAGCCTGACGC

-   -   a region (italic bold upper case) for addition of a BstZ17I,        EcoRI and BglII restriction sites    -   a region (underlined lower case) for addition of a Ribosome        Binding Site    -   a region (upper case) homologous to the E. coli MG1655 yeaB        region from 1894195 to 1894215 (reference sequence on the        EcoGene website),

yeaB R (SEQ ID NO 73) GCTTATAAGCCAGCTGGCTCTAGATAGCAGAAAGGCCCACCCGAAGGTGAGCCAGGA GTATACATGAAGCATTTCCGTTAATTAACGGAGCTCATC CTAGGTCAGGGTTTCACACCAATTTGCAGCGCC

-   -   a region (bold upper case) homologous to the E. coli MG1655 yeaB        region from 1894772 to 1894745 (reference sequence on the        EcoGene),    -   a region (underlined upper case) homologous to the terminator        sequence T7Te (ref: Harrington K. J., Laughlin R. B. and        Liang S. Proc Natl Acad Sci USA. 2001 Apr. 24; 98(9):5019-24):    -   a region (italic upper case) for addition of a PsiI, PvuII,        XbaI, BstZ17I, XmnI, PacI, SacI and AvrII restriction sites

The PCR amplified fragment was cut with the restriction enzymes XbaI andBglII and cloned into the XbaI-BglII sites of the vectorpME101-kivDll-yqhD-TT07 giving vector pME101-kivDll-yqhD-yeaB-TT07.

6.2 Construction of a Plasmid for Overexpression of the PhosphoglycerateDehydrogenase serA of Escherichia coli: pCC1BAC-serA Plasmid

To increase the expression of the serA gene, the gene was expressed fromthe copy control vector pCC1BAC (Epicentre) using its proper promoter.

For this purpose, the serA gene was amplified from the E. coli genomeusing the oligonucleotides serA F and serA R. The PCR product wasrestricted using enzymes XbaI and SmaI and cloned into the vector pUC18(Stratagene) restricted by the same restriction enzymes. The resultingvector was named pUC18-serA.

serA F (SEQ ID NO 74):

ctagTCTAGATTAGTACAGCAGACGGGCGCG

with

-   -   a region (upper case) homologous to the sequence        (3055199-3055220) of the gene serA (reference sequence on the        EcoGene website),    -   a region (bold case) harbouring the XbaI site

serA R (SEQ ID NO 75):

tccCCCGGGAAGCTTCCGTCAGGGCGTGGTGACCG

with

-   -   a region (upper case) homologous to the sequence        (3056880-3056861) of the gene serA region (reference sequence on        the EcoGene website),    -   a region (bold case) harbouring the SmaI and HindIII sites

To transfer the gene serA into the copy control vector pCC1BAC, thevector pUC18-serA was restricted with the enzyme HindIII and cloned intoHindIII cloning ready pCC1BAC (Epicentre).

The resulting construct was verified and called pCC1BAC-serA.

6.3 Construction of the MG1655 ΔpykF (pME101-kivDll-yqhD-yeaB-TT07)(pCC1BAC-serA) Strain

The MG1655 ΔpykF strain construction was previously detailed (part 2.2)

The pCC1BAC-serA and the pME101-kivDll-yqhD-yeaB-TT07 plasmids were thenintroduced into the strain MG1655 ΔpykF.

Example 7 Demonstration of the Hydroxy Keto-Acid Decarboxylase ActivityEncoded by the Gene kivD of Lactococcus lactis

7.1 Construction of Strain for KivD Characterisation: BL21(pPAL7-kivDll)

To characterise the KivD protein, the corresponding gene was expressedfrom the expression vector pPAL7 (Bio-rad).

For this purpose, the kivD gene was amplified from the Lactococcuslactis genome using the oligonucleotides pPAL7-kivDll F and pPAL7-kivDllR. The PCR product was restricted using enzymes HindIII and EcoRI andcloned into the vector pPAL7 restricted by the same restriction enzymes.The resulting vector was named pPAL7-kivDll.

pPAL7-kivDll F (SEQ ID NO 76):

cccAAGCTTtgACTTCTATGTATACCGTGGGTGATTATC

with

-   -   a region (italic case) homologous to the sequence of the        synthetic gene of the Lactococcus lactis kivD gene,        -   a region (bold case) harbouring the nucleotides necessary to            generate tag-free protein containing a short N-terminal            amino acid extension to favour the purification        -   a region (underlined case) harbouring the HindIII            restriction site pPAL7-kivDll R (SEQ ID NO 77):

gGAATTCTTAGCTTTTATTCTGTTCGGCGAACAG

with

-   -   a region (italic case) homologous to the sequence of the        synthetic gene of the Lactococcus lactis kivD gene,    -   a region (underlined case) harbouring the EcoRI restriction site

The pPAL7-kivDll plasmid was then introduced into the strain BL21 (DE3)competent cells (Invitrogen).

7.2 Overproduction of the Protein KivD

The overproduction of the protein KivD was done in a 2 l Erlenmeyerflask, using LB broth (Bertani, 1951, J. Bacteriol. 62:293-300) that wassupplemented with 2.5 g/l glucose and 100 mg/l of ampicillin. Anovernight preculture was used to inoculate a 500 ml culture to anOD_(600 nm) of about 0.15. This preculture was carried out in a 500 mlErlenmeyer flask filled with 50 ml of LB broth that was supplementedwith 2.5 g/l glucose and 100 mg/l of ampicillin. The culture was firstkept on a shaker at 37° C. and 200 rpm until OD_(600 nm) was about 0.5and then the culture was moved on a second shaker at 25° C. and 200 rpmuntil OD_(600 nm) was 0.6-0.8 (about one hour), before induction with500 μM IPTG. The culture was kept at 25° C. and 200 rpm untilOD_(600 nm) was around 4, and then it was stopped. Cells werecentrifuged at 7000 rpm, 5 minutes at 4° C., and then stored at −20° C.

7.3 Purification of the Protein KivD

7.3.1 Step 1: Preparation of Cell-Free Extracts.

About 188 mg of E. coli biomass was suspended in 30 ml of 100 mMpotassium phosphate pH 7.6, and a protease inhibitor cocktail. The cellsuspension (15 ml per conical tube) was sonicated on ice (Bandelinsonoplus, 70 W) in a 50 ml conical tube during 8 cycles of 30 sec with30 sec intervals. After sonication, cells were incubated for 30 min atroom temperature with 5 mM MgCl2 and 1UI/ml of DNaseI. Cells debris wasremoved by centrifugation at 12000 g for 30 min at 4° C.

7.3.2 Step 2: Affinity Purification

The protein was purified from crude cell-extract by affinity on aProfinity column (BIORAD, Bio-Scale Mini Profinity exact cartridge 5 ml)according to the protocol recommended by the manufacturer. Crude extractwas loaded on a 5 ml Profinity exact cartridge equilibrated with 100 mMpotassium phosphate pH 7.6. The column was washed with 10 column volumesof the same buffer and incubated overnight with 100 mM potassiumphosphate pH 7.6, 100 mM fluoride at 4° C. The protein was eluted fromthe column with 2 column volumes of 100 mM potassium phosphate pH 7.6.The tag remained tightly bound to the resin and the purified protein wasreleased. The fractions containing the protein were pooled and dialyzedagainst 100 mM potassium phosphate, 150 mM NaCl and 10% glycerol pH 8.

Protein concentration was measured using the Bradford protein assay.

7.4 Hydroxy Keto-Acid Decarboxylase Assay

7.4.1 Chemical synthesis of 5-hydroxy-2-ketopentanoic acid

Chemical synthesis of 5-hydroxy-2-ketopentanoic acid has been describedin the publication:

Friedhelm Korte, Karl Heinz Büchel,α-Hydroxyalkyliden-lacton-Umlagerung, X.α-Hydroxyalkyliden-lacton-Umlagerung in wäβriger Salzsäure ChemischeBerichte, Volume 92 Issue 4, Pages 877-883 (1959)

7.4.2 Chemical Synthesis of 4-hydroxy-2-ketobutyric Acid

Chemical synthesis of 4-hydroxy-2-ketobutyric acid has been described inthe publication: R S Lane; EE Dekker; (1969). 2-keto-4-hydroxybutyrate.Synthesis, chemical properties, and as a substrate for lactatedehydrogenase of rabbit muscle Biochemistry., 8 (7), 2958-2966.

7.4.3 Hydroxy Keto-Acid Decarboxylase Assay

The decarboxylation of hydroxy keto-acids was measured at 30° C. using acoupled enzymatic assay. The hydroxy keto-acid decarboxylase activityassay was carried out with 50 mM potassium phosphate buffer pH 6, 0.2 mMNADH, 1 mM MgSO4, 0.5 mM thiamin diphosphate, 72 units/ml alcoholdehydrogenase from Saccharomyces cerevisiae, 10 mM hydroxy keto-acidneutralized (Hydroxypyruvic acid or 4-hydroxy-2-ketobutyric acid or5-hydroxy-2-ketopentanoic acid) and about 40 μg of purified protein in atotal volume of 1 ml. The consumption of NADH was monitored at 340 nm ona spectrophotometer. The activity detected in control assay, lacking thesubstrate, was subtracted from the activity detected in the assay withsubstrate. A unit of hydroxy keto-acid decarboxylase activity is theamount of enzyme required to catalyze the decarboxylation of 1 mmol ofhydroxy keto-acid per min at 30° C. (Epsilon 340 nm=6290 M−1 cm−1)

7.5 Activity of Purified Enzyme

Activity of purified enzyme (mUI/mg) Hydroxypyruvate decarboxylase assay79 4-hydroxy-2-ketobutyrate decarboxylase assay 705-hydroxy-2-ketopentanoate decarboxylase assay 63

Example 8 Demonstration of the Hydroxy Aldehyde Reductase ActivityEncoded by the Gene yqhD of Escherichia coli

8.1 Construction of a Strain for yqhD Characterisation: MG1655 ΔpykF::Km(pTRC99A-yqhD)

8.1.1 Construction of Strain MG1655 ΔyqhD::Km

To delete the yqhD gene, the homologous recombination strategy describedby Datsenko & Wanner (2000) was used. This strategy allows the insertionof a chloramphenicol or a kanamycin resistance cassette, while deletingmost of the genes concerned. For this purpose the followingoligonucleotides were used:

ΔyqhDF (SEQ ID NO 78)

atgaacaactttaatctgcacaccccaacccgcattctgrttggtaaaggcgcaatcgctggtttacgcgaacaaattccgtgtaggctggagctgcttcg

with

-   -   a region (lower case) homologous to the sequence (3153377        to 3153456) of the yqhD region (reference sequence on the        EcoGene website),    -   a region (upper case) for the amplification of the kanamycin        resistance cassette (reference sequence in Datsenko, K. A. &        Wanner, B. L., 2000, PNAS, 97: 6640-6645),

ΔyqhDR (SEQ ID NO 79)

ttagcgggcggcttcgtatatacggcggctgacatccaacgtaatgtcatgattttcgcccagttgggtcatgccgtgctcccatatgaatatcctccttag

with

-   -   a region (upper case) homologous to the sequence (3154540        to 3154460) of the yqhD region (reference sequence on the        EcoGene website),    -   a region (upper case) for the amplification of the kanamycin        resistance cassette (reference sequence in Datsenko, K. A. &        Wanner, B. L., 2000, PNAS, 97: 6640-6645).

The oligonucleotides ΔyqhDF and ΔyqhDR are used to amplify the kanamycinresistance cassette from the plasmid pKD4. The PCR product obtained isthen introduced by electroporation into the strain MG 1655 (pKD46). Thekanamycin resistant transformants are then selected and the insertion ofthe resistance cassette is verified by a PCR analysis with theoligonucleotides yqhDF and yqhDR defined below. The strain retained isdesignated MG1655 ΔyqhD::Km. yqhDF (SEQ ID NO 80):ggcgtctcgccatacaacaaacgcacatcgggc (homologous to the sequence from3153068 to 3153100). yqhDR (SEQ ID NO 81): gggctttgccgacaccttcttcgttcttg(homologous to the sequence from 3154825 to 3154797).

8.1.2 Construction of Plasmid pTRC99A-yqhD

To characterise the YqhD protein, the corresponding gene was expressedfrom the vector pTRC99A (Amersham).

For this purpose, the yqhD gene was amplified from the E. coli genomeusing the oligonucleotides yqhD F pTRC99A F and yqhD R pTRC99A R. ThePCR product was restricted using enzymes HindIII and BspHI and clonedinto the vector pTRC99A restricted by the NcoI-HindIII restrictionenzymes. The resulting vector was named pTRC99A-yqhD.

yqhD F pTRC99A F (SEQ ID NO 82):

cgatgcacgtcatgaacaactttaatctgcacaccccaacccg,

with:

-   -   a region (underlined case) homologous to the sequence (3153377        to 3153408) of the gene yqhD (reference sequence on the EcoGene        website),    -   a BspHI restriction site (bold case)

yqhD R pTRC99A R (SEQ ID NO 83):

ggcgtaaaaagcttagcgggcggcttcgtatatacggcggctgacatccaacgtaatgtcgtgattttcg

with:

-   -   a region (underlined case) homologous to the sequence (3154540        to 3154483) of the gene yqhD (reference sequence on the EcoGene        website),    -   a HindIII restriction site (bold case)

The pTRC99A-yqhD plasmid was then introduced into the strain MG1655ΔyqhD::Km.

8.2 Overproduction of the Protein YqhD

The protein YqhD was overproduced at 37° C. under aerobic conditions in2 l baffled Erlenmeyer flasks with 500 ml LB medium with 2.5 g/l glucoseand 50 mg/l of ampicillin and 50 mg/l of kanamycin. The flasks wereagitated at 200 rpm on an orbital shaker. When the optical densitymeasured at 550 nm reached 0.5 units, the flasks were incubated at 25°C. When the optical density reached 1.2 units, the production of YqhDproteins was induced by adding IPTG to a final concentration of 500 μM.The biomass was harvested by centrifugation when the cultures reached anoptical density above 3.5 units. The supernatant was discarded and thepellet was stored at −20° C. before use.

8.3 Purification of the Protein YqhD

8.3.1 Step 1: Preparation of Cell-Free Extracts.

400 mg of E. coli biomass were suspended in 70 ml of 50 mM Hepes pH 7.5,and a protease inhibitor cocktail. Cells were sonicated on ice (Bransonsonifier, 70 W) in a Rosett cell RZ3 during eight cycles of 30 sec with30 sec intervals. After sonication, cells were incubated for 1 hour atroom temperature with 1 mM MgCl2 and 1 UI/ml of DNaseI. Cells debris wasremoved by centrifugation at 12000 g for 30 min at 4° C. The supernatantwas kept as the crude extract.

8.3.2 Step 2: Ammonium Sulphate Precipitation

The crude extract was precipitated at a concentration of 50% ammoniumsulphate: solid ammonium sulphate (300 g/l) was added to the crudeextract on ice. After 15 min of incubation at 4° C., the mix wascentrifuged at 12000 g for 15 min at 4° C. The supernatant was discardedand the precipitate dissolved in 50 ml of 50 mM Hepes pH 7.5, 1 Mammonium sulphate.

8.3.3 Step 3: Hydrophobic Chromatography.

Using an Akta Purifier (GE Healthcare), the protein extract from theprevious step was loaded onto a 5 ml HiTrap PhenylHP column (GEHealthcare) equilibrated with the same buffer. The column was washedwith 10 column volumes of the same buffer. Proteins were eluted with twostep gradients, a gradient of 10 column volumes from 1 M to 0.5 Mammonium sulphate and a gradient of 20 column volumes from 0.5 M to 0 Mammonium sulphate. After elution, the column was washed with 10 columnvolumes of 50 mM Hepes pH 7.5. The flow rate of the column was 2.5ml/min and 2.5 ml fractions were collected.

The fractions which contain the protein were pooled, dialyzed in 50 mMHepes pH 7.5 and concentrated to a concentration of 1.14 μg/μl.

8.4 Hydroxy Aldehyde Reductase Assays

8.4.1 Chemical synthesis of 4-hydroxybutyraldehyde

Chemical synthesis of 4-hydroxybutyraldehyde has been described in thepublication: No 158 Transposition des dihydro-2.5 furannes endihydro-2.3 furannes. —Application àla préparation de l'hydroxy-4butanal; par R. PAUL, M. FLUCHAIRE et G. GOLLARDEAU.

Bulletin de la Société Chimique de France, 668-671, 1950.

8.4.2 Glycolaldehyde and 4-hydroxybutyraldehyde Reductase Activity Assay

Glycolaldehyde and 4-hydroxybutyraldehyde reductase activity was assayedby measuring the initial rate of NADPH oxidation with aspectrophotometer at a wavelength of 340 nm and at a constanttemperature of 30° C. The reaction mixture using glycolaldehyde or4-hydroxybutyraldehyde as substrate was carried out in 20 mM Hepes pH7.5, 0.1 mM Zinc sulphate, 0.2 mM NADPH, 2 μg of purified enzyme in afinal volume of 1 ml. The reaction mixture was incubated for 5 min at30° C. and then the reaction was initiated by the addition of thesubstrate (glycolaldehyde or 4-hydroxybutyraldehyde) at a finalconcentration of 10 mM. A control assay (blank), lacking the substrate,was run in parallel and the value measured for the control wassubtracted from the value measured for the assay to take into accountnon-specific oxidation of NADPH. (Epsilon 340 nm=6290 M−1 cm−1).

One unit of enzyme activity was defined as the amount of enzyme thatconsumed 1 μmol substrate per minute under the conditions of the assay.Specific enzyme activity was expressed as units per mg of protein.

8.4.3 3-hydroxypropionaldehyde Reductase Activity Assay

The activity of YqhD toward the substrate 3-hydroxypropionaldehyde(3-HPA) has been described in the publication:

-   Hongmei Li; Jia Chen; Hao Li; Yinghua Li; Ying Li; (2008). Enhanced    activity of yqhD oxidoreductase in synthesis of 1,3-propanediol by    error-prone PCR Prog Nat. Sci., 18 (12), 1519-1524.

These authors have used 5 mM Zinc chloride, 1 mM EDTA and 1 mMBeta-mercaptoethanol for their 3-hydroxypropionaldehyde reductaseassays.

8.5 Activity of Purified Enzyme

Activity of purified enzyme (mUI/mg) Glycoladehyde reductase activityassay 9840 3-hydroxypropionaldehyde reductase activity assay 79184-hydroxybutyraldehyde reductase activity assay 1443

Example 9 Demonstration of the L-Serine Transaminase and L-HomoserineTransaminase Activity Encoded by the Gene serC of Escherichia coli

9.1 Construction of Strain for SerC Characterisation: BL21 (pPAL7-serC)

To characterise the SerC protein, the corresponding gene was expressedfrom the expression vector pPAL7 (Bio-rad).

For this purpose, the serC gene was amplified from the E. coli genomeusing the oligonucleotides pPAL7-serC F and pPAL7-serC R. The PCRproduct was restricted using enzymes HindIII and EcoRI and cloned intothe vector pPAL7 restricted by the same restriction enzymes. Theresulting vector was named pPAL7-serC.

pPAL7-serC F (SEQ ID NO 84):

cccAAGCTTtgATGGCTCAAATCTTCAATTTTAGTTCTGG

with

-   -   a region (bold case) homologous to the sequence (956876-956904)        of the gene serC (reference sequence on the EcoGene website),    -   a region (underlined case) harbouring the HindIII restriction        site pPAL7-serC R (SEQ ID NO 85):

gGAATTCTTAACCGTGACGGCGTTCGAACTCAACC

with

-   -   a region (bold case) homologous to the sequence (957964-957937)        of the gene serC region (reference sequence on the EcoGene        website),    -   a region (underlined case) harbouring the EcoRI restriction site

The pPAL7-serC plasmid was then introduced into competent BL21 (DE3)cells (Invitrogen).

9.2 Overproduction of the Protein SerC

The overproduction of the protein SerC was done applying the sameprotocol as example #7.2

9.3 Purification of the Protein SerC

9.3.1 Step 1: Preparation of Cell-Free Extracts

About 280 mg of E. coli biomass was suspended in 45 ml of 100 mMpotassium phosphate pH 7.6, and a protease inhibitor cocktail. The cellsuspension (15 ml per conical tube) was sonicated on ice (Bandelinsonoplus, 70 W) in a 50 ml conical tube during 8 cycles of 30 sec with30 sec intervals. After sonication, cells were incubated for 30 min atroom temperature with 5 mM MgCl2 and 1 UI/ml of DNaseI. Cells debris wasremoved by centrifugation at 12000 g for 30 min at 4° C.

9.3.2 Step 2: Affinity Purification

The protein was purified from the crude cell-extract by affinity on aProfinity column (BIORAD, Bio-Scale Mini Profinity exact cartridge 5 ml)according to the protocol recommended by the manufacturer. The crudeextract was loaded on a 5 ml Profinity exact cartridge equilibrated with100 mM potassium phosphate pH 7.6. The column was washed with 10 columnvolumes of the same buffer and incubated 30 min with 100 mM potassiumphosphate pH 7.6, 100 mM fluoride at room temperature. The protein waseluted from the column with 2 column volumes of 100 mM potassiumphosphate pH 7.6. The tag remained tightly bound to the resin and thepurified protein was released. The fractions containing the protein werepooled and dialyzed against 100 mM Tris HCl, 150 mM NaCl and 10%glycerol pH 8.

Protein concentration was measured using the Bradford protein assay.

9.4 L-Serine Transaminase Activity Assay

For the L-serine transaminase activity assay about 30 μg of purifiedenzyme was added to a buffer containing 50 mM Tris-HCl buffer pH 8.2, 3mM L-Serine, 1 mM α-ketoglutaric acid in a total volume of 300 μl. Thereaction was incubated during 60 min at 30° C. The reaction product(hydroxypyruvic acid) was measured directly by LC-MS/MS.

9.5 L-homoserine Transaminase Assay

The L-homoserine transaminase activity was measured at 30° C. using acoupled enzymatic assay. The L-homoserine transaminase activity assaywas carried out with 420 mM potassium phosphate buffer pH 8.2, 2 mMacetylpyridine adenine dinucleotide, 3 mM L-homoserine, 20 units/mlglutamic dehydrogenase from bovine liver, 1 mM alpha-ketoglutaric acidneutralized and about 50 μg of crude extract in a total volume of 1 ml.The consumption of acetylpyridine adenine dinucleotide was monitored at375 nm on a spectrophotometer. The activity detected in control assay,lacking the substrate (L-homoserine), was subtracted from the activitydetected in the assay with substrate. A unit of L-homoserinetransaminase activity is the amount of enzyme required to catalyze thetransamination of 1 μmol of L-homoserine per min at 30° C. (Epsilon 375nm=6100 M−1 cm−1)

9.6 Activities of Purified Enzyme

Activity of purified enzyme (mUI/mg) L-Serine transaminase assay 186L-Homoserine transaminase assay 118

Example 10 Demonstration of the 3-phosphohydroxypyruvate PhosphataseActivity Encoded by the Gene GPP2 of Saccharomyces cerevisiae

10.1 Construction of a Strain for GPP2sc Characterization: BL21(pPAL7-gpp2sc)

To characterise the GPP protein, the corresponding gene is expressedfrom the expression vector pPAL7 (Bio-rad).

For this purpose, the gpp gene is amplified from the Saccharomycescerevisiae genome using the oligonucleotides pPAL7-gpp2sc F andpPAL7-gpp2sc R. The PCR product is restricted using enzymes HindIII andBamHI and cloned into the vector pPAL7 restricted by the samerestriction enzymes. The resulting vector is named pPAL7-gpp2sc.pPAL7-gpp2sc F (SEQ ID NO 86):

cccAAGCTTTgATGGGATTGACTACTAAACCTCTATC

with

-   -   a region (bold case) homologous to the sequence (280680-280655)        of the gene gpp2 region (reference sequence on the Saccharomyces        Genome Database website),    -   a region (underlined case) harbouring the HindIII restriction        site pPAL7-kivDll R (SEQ ID NO 87):

gGGATCCTTACCATTTCAACAGATCGTCCTTAGC

with

-   -   a region (bold case) homologous to the sequence (279928-279954)        of the gene gpp2 region (reference sequence on the Saccharomyces        Genome Database website),    -   a region (underlined case) harbouring the BamHI restriction site

The pPAL7-gpp2sc plasmid is then introduced into competent BL21 (DE3)cells (Invitrogen).

10.2 Overproduction of the Protein GPP2sc

The overproduction of the protein GPP2sc was done applying the sameprotocol as example #7.2

10.3 Purification of the Protein GPP2sc

10.3.1 Step 1: Preparation of Cell-Free Extracts.

About 294 mg of E. coli biomass was suspended in 45 ml of 100 mMpotassium phosphate pH 7.6, and a protease inhibitor cocktail. The cellsuspension (15 ml per conical tube) was sonicated on ice (Bandelinsonoplus, 70 W) in a 50 ml conical tube during 8 cycles of 30 sec with30 sec intervals. After sonication, cells were incubated for 30 min atroom temperature with 5 mM MgCl2 and 1 UI/ml of DNaseI. Cells debris wasremoved by centrifugation at 12000 g for 30 min at 4° C.

10.3.2 Step 2: Affinity Purification

The protein was purified from the crude cell-extract by affinity on aProfinity column (BIORAD, Bio-Scale Mini Profinity exact cartridge 5 ml)according to the protocol recommended by the manufacturer. The crudeextract was loaded on a 5 ml Profinity exact cartridge equilibrated with100 mM potassium phosphate pH 7.6. The column was washed with 10 columnvolumes of the same buffer and incubated overnight with 100 mM potassiumphosphate pH 7.6, 100 mM fluoride at room temperature. The protein waseluted from the column with 2 column volumes of 100 mM potassiumphosphate pH 7.6.

The tag remained tightly bound to the resin and the purified protein wasreleased. The fractions containing the protein were pooled and dialyzedagainst 100 mM potassium phosphate, 150 mM NaCl, 10% glycerol pH 8 andconcentrated to a concentration of 0.22 μg/μl.

Protein concentration was measured using the Bradford protein assay.

10.4 3-phosphohydroxypyruvate Phosphatase Activity Assay

10.4.1 Chemical synthesis of 3-phosphohydroxypyruvate

Chemical synthesis of 3-phosphohydroxypyruvate has been described in thepublication: C E Ballou; H Hesse; R Hesse; (1956). The Synthesis andProperties of Hydroxypyruvic Acid Phosphate J Am Chem. Soc., 78 (15),3718-3720.

10.4.2 3-Phosphohydroxypyruvate Phosphatase Activity Assay

3-phosphohydroxypyruvate phosphatase activity assay was carried out with50 mM Tris-HCl buffer pH 8.2, 5 mM MgCl₂, 3.6 mM3-phosphohydroxypyruvate and about 6 μg of purified enzyme (Gpp) in atotal volume of 300 μl. The reaction was incubated during 120 min at 30°C. The reaction product (hydroxypyruvic acid) was measured directly byLC-MS/MS.

10.5 Activity of Purified Enzyme

Activity of purified enzyme (mUI/mg) 3-phosphohydroxypyruvatephosphatase assay 9

Example 11 Simulation of Maximum Yields for Ethylene Glycol,1,3-propanediol and 1,4-butanediol Production

11.1 Parameters Used for Simulations

Simulations have been performed with our METEX proprietary softwareMETOPT™. A simplified metabolic network of E. coli has been usedincluding a central metabolic network, metabolic pathways for allbiomass precursors and specific production pathways as described above.A classical biomass composition for E. coli has been used. For eachspecific diol, two simulations have been performed. The first one tocalculate a theoretical maximum yield (taking into account onlystoichiometry of the model, with no growth and no maintenance energy).The second one to calculate a practical maximum yield, taking intoaccount a growth rate of 0.1 h⁻¹ and a maintenance energy of 5mmol_(ATP)·g_(DW) ⁻¹·h⁻¹. All simulations have been performed with aspecific uptake rate of glucose of 3 mmol·g_(DW) ⁻¹·h⁻¹. For ethyleneglycol and 1,3-propanediol, simulations have been performed in aerobicconditions. Specifically for 1,4-butanediol, simulations have beenperformed both in aerobic and anaerobic conditions. For anaerobicconditions, the growth rate could not be imposed to 0.1 h⁻¹. The growthrate is the maximal growth rate attainable according to available ATP.

11.2 Simulations Results

Ethylene 1,3- 1,4-butanediol 1,4-butanediol glycol propanediol (aerobic)(anaerobic) Maximum 0.69 0.60 0.50 0.50 theoretical yield (g/g) Maximum0.50 0.39 0.35 0.45 practical yield (μ_(max) = 0.03 h⁻¹) (g/g)

Example 12 Construction of Strains with Increased 1.3-propanediolPathway Flux and Expressing a 2-keto Acid Decarboxylase Encoding Geneand an Hydroxy Aldehyde Reductase Encoding Gene MG1655 ΔpykF ΔmetAΔthrLABC (pBBR1MCS5-Ptrc01/RBS01*2-yqhD-kivDll-TT07)(pME101-thrA*1-serC)

12.1 Construction of Strain MG1655 ΔpykF

To delete the pykF gene, the homologous recombination strategy describedby Datsenko and Wanner (2000, PNAS, 97: 6640-6645) was used. Theconstruction was performed as described in the previous example 2.2

The kanamycin resistance cassette was eliminated. The plasmid pCP20carrying FLP recombinase acting at the FRT sites of the kanamycinresistance cassette was then introduced into the recombinant sites byelectroporation. After a series of cultures at 42° C., the loss of thekanamycin resistance cassettes was verified by a PCR analysis with thesame oligonucleotides as used previously (pykFF/pykFR). The strainretained was designated MG1655 ΔpykF.

12.2 Construction of Strain MG1655 ΔpykF ΔmetA

To delete the metA gene, the homologous recombination strategy describedby Datsenko & Wanner (2000) was used. The construction was performed asdescribed in the previous example 3.1.

The strain retained was designated MG1655 ΔpykF ΔmetA.

12.3 Construction of Strain MG1655 ΔpykF ΔmetA ΔthrLABC

To delete the thrLABC operon, the homologous recombination strategydescribed by Datsenko & Wanner (2000) was used. This strategy allows theinsertion of a chloramphenicol or a kanamycin resistance cassette, whiledeleting most of the genes concerned. For this purpose the followingoligonucleotides were used. DthrLABF (SEQ ID NO 88:

cgggcaatatgtctctgtgtggattaaaaaaagagtgtctgatagcagcttctgaactggttaccttcctggctcaccttcgggtgggcctttctggtatacTGTAGGCTGGAGCTGCTTCG with

-   -   a region (lower case) homologous to the sequence (22-86) of the        thrLABC region (reference sequence on the EcoGene website),    -   a region (bold underlined lower case) for T7Te transcriptional        terminator sequence from T7 phage (Harrington K. J.,        Laughlin R. B. and Liang S. Proc Natl Acad Sci USA. 2001 Apr.        24; 98(9):5019-24),    -   a region (upper case) for the amplification of the        chloramphenicol resistance cassette (reference sequence in        Datsenko, K. A. & Wanner, B. L., 2000, PNAS, 97: 6640-6645),        DthrLABCR (SEQ ID NO 89):

CCCTGTCATTTTTCTCCATAATTTCTTCATAAAAAAGCCGGGCTGCATAAAAGCAAACCCGGCCTGATTGAGATAATGAATAGATTCCCGGGGGAGGCGCCCGCGGATCCCATATGAATATCCTCCTTAG

with

-   -   a region (upper case) homologous to the sequence (5106-5021) of        the thrLABC region (reference sequence on the EcoGene website),    -   a region (italic upper case) for addition of a BamHI-SfoI-SmaI        restriction sites    -   a region (bold upper case) for the amplification of the        chloramphenicol resistance cassette.

The oligonucleotides ΔthrBF and ΔthrCR were used to amplify thechloramphenicol resistance cassette from the plasmid pKD3. The PCRproduct obtained was then introduced by electroporation into the strainMG1655 (pKD46). The chloramphenicol resistant transformants were thenselected and the insertion of the resistance cassette was verified by aPCR analysis with the oligonucleotides thrLF and thrCR defined below.The strain retained was designated MG1655 ΔthrLABC::Cm.

thrLF (SEQ ID NO 90): GCCATGCCGCGCTGGTGTTTGGTCGCG (homologous to thesequence from 4639281 to 4639307). thrCR (SEQ ID NO 91):GCGACCAGAACCAGGGAAAGTGCG (homologous to the sequence from 5283 to 5260).

To transfer the ΔthrLABC::Cm, the method of phage P1 transduction wasused. The preparation of the phage lysate of the strain MG1655ΔthrLABC::Cm was used for the transduction into the strain MG1655 ΔpykFΔmetA. The chloramphenicol resistant transformants were then selectedand the ΔthrLABC::Cm was verified by a PCR analysis with the previouslydefined oligonucleotides thrLF and thrCR. The strain retained wasdesignated MG1655 ΔpykF ΔmetA ΔthrLABC::Cm.

The chloramphenicol resistance cassette was eliminated. The plasmidpCP20 carrying FLP recombinase acting at the FRT sites of thechloramphenicol resistance cassettes was then introduced into therecombinant sites by electroporation. After a series of cultures at 42°C., the loss of the chloramphenicol resistance cassette was verified bya PCR analysis with the same oligonucleotides as used previously((pykFF/pykFR, metAF/metAR, and thrLF/thrCR). The strain retained wasdesignated MG 1655 ΔpykF ΔmetA ΔthrLABC.

12.4 Construction of a Plasmid for Overexpression of the L-homoserineTransaminase serC of Escherichia coli: pME101-thrA*1-serC Plasmid

To increase the expression of the serC gene, the gene was expressed fromthe pME101-thrA*1 previously described (PCT_WO2008707041) using itsproper promoter.

For this purpose, the serC gene was amplified from the E. coli genomeusing the oligonucleotides serC F and serC R. The PCR product wasrestricted using enzymes XbaI and SmaI and cloned into the vectorpME101-thrA*1 restricted by the same restriction enzymes. The resultingvector was named pME101-thr A*1-serC.

serC F (SEQ ID NO 92):

TGCTCTAGAGTCCGCGCTGTGCAAATCCAGAATGG

with

-   -   a region (upper case) homologous to the sequence (956619-956644)        of the gene serC (reference sequence on the EcoGene website),    -   a region (bold upper case) harbouring the XbaI site serC R (SEQ        ID NO 93):

CCCAAGCTTAACTCTCTACAACAGAAATAAAAAC

with

-   -   a region (upper case) homologous to the sequence (958028-958004)        of the gene serC region (reference sequence on the EcoGene        website),    -   a region (bold upper case) harbouring the HindIII site

The PCR amplified fragment was cut with the restriction enzymes XbaI andHindIII and cloned into the XbaI-HindIII sites of the vectorpME101-thrA*1 giving vector pME101-thrA*1-serC.

12.5 Construction of a Plasmid for the Overexpression of the HydroxyAldehyde Reductase yqhD Gene of Escherichia coli and theAlpha-Ketoisovalerate Decarboxylase kivD Gene of Lactococcus lactis:pBBR1MCS5-Ptrc01/RBS01*2-yqhD-kivDll-TT07 Plasmid

The pME101-yqhD-kivDll-TT07 plasmid was first constructed. The kivDllgene from the pME101-kivDll-TT07 vector (PCT/2009/067994) restricted byBsrBI and BglII was cloned into the pME101VB01-yqhD vector (previouslydescribed in PCT/2007/000509) restricted by SnaBI and BglII, theresulting plasmid was named pME101-yqhD-kivDll-T07. The yqhD and kivDllgenes were then PCR amplified from the pME101-yqhD-kivDll-T07 plasmidwith the oligonucleotides Ptrc01-RBS01-yqhD pBBR F and kivD pBBR R. ThePCR product was digested with the restriction enzymes SpeI and SmaI andcloned into the vector pBBR1MCS5 (M. E. Kovach, (1995), Gene166:175-176) restricted by the same enzymes, giving thepBBR1MCS5-Ptrc01/RBS01*2-yqhD-kivDll-TT07 vector.

Ptrc01-RBS01-yqhD pBBR F (SEQ ID NO 94)

AgaACTAGTgagctgttgacaattaatcatccggctcgtataatgtgtggaagtcgacGGATCCtaaggaggttataaatgaacaactttaatctgcacacccc

-   -   a region (bold upper case) for addition of a SpeI restriction        site    -   a region (bold lower case) for addition of the constitutive Ptrc        promoter sequence    -   a region (italic upper case) for addition of a BamHI restriction        site    -   a region (underlined lower case) for addition of the Ribosome        Binding Site sequence    -   a region (italic lower case) homologous to the sequence        (3153377-3153402) of the MG1655 yqhD gene (on the EcoGene        website)

kivD pBBR R (SEQ ID NO 95)

GAGCCCGGGGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGATACGTAGAATTCTTAATTAAGTTAGCTTTTATTCTGTTCGGCG

-   -   a region (bold italic upper case) for addition of a SmaI        restriction site    -   a region (underlined upper case) for T7Te transcriptional        terminator sequence from T7 phage (Harrington K. J.,        Laughlin R. B. and Liang S. Proc Natl Acad Sci USA. 2001 Apr.        24; 98(9):5019-24),    -   a region (bold upper case) for addition of a SnaBI-EcoRI-PacI        restriction sites    -   a region (italic upper case) homologous to the end of the        synthetic kivD gene

12.6 Construction of Strain MG1655 ΔpykF ΔmetA ΔthrLABC(pME101-thrA*1-serC) (pBBR1MCS5-Ptrc01/RBS01*2-yqhD-kivDll-TT07)

The pME101-thrA*1-serC and pBBR1MCS5-Ptrc01/RBS01*2-yqhD-kivDll-TT07plasmids were then introduced into the strain MG1655 ΔmetA ΔpykFΔthrLABC.

12.7 Culture for 1,3-propanediol Production

Performances of strains were assessed in 500 ml baffled Erlenmeyer flaskcultures using modified M9 medium (Anderson, 1946, Proc. Natl. Acad.Sci. USA 32:120-128) that was supplemented with 4.5 mM threonin, 5 mMmethionin, 10 g/l MOPS and 10 g/l glucose and adjusted at pH 6.8.Spectinomycin and gentamycin were added at a concentration of 50 mg/l.100 μM IPTG was also added for induction of the expression vectorpME101. A 24 hours preculture was used to inoculate a 50 ml culture toan OD_(600 nm) of about 0.1. The cultures were kept on a shaker at 37°C. and 200 rpm until the glucose in the culture medium was exhausted. Atthe end of the culture, glucose and major products were analyzed by HPLCusing a Biorad HPX 97H column for the separation and a refractometer forthe detection. Production of 1,3-propanediol was confirmed by LC/MS/MS.

The performances of different strains are given in table below.

[1,3-PDO] Culture_ref Strain_ref Genotype (mM) FbDI335 DI0084c02 MG1655ΔpykF ΔmetA nd FbDI340 ΔthrLABC FbDI357 FbDI336 DI0107c01 (MG1655 ΔpykFΔmetA 0.391 +/− 0.125 FbDI341 ΔthrLABC (pME101-thrA FbDI358 *1-serC)(pBBR1MCS5- FbDI395 Ptrc01/RBS01*2-yqhD- kivDll-TT07) nd: not detected

The invention claimed is:
 1. An E. coli microorganism geneticallymodified for the bioproduction of an aliphatic diol, wherein themicroorganism comprises a metabolic pathway for the decarboxylation of ahydroxy-2-keto-aliphatic acid metabolite with an enzyme having a 2-ketoacid decarboxylase activity encoded by the gene kivD from L. lactis, theproduct obtained from said decarboxylation step being further reducedinto the corresponding aliphatic diol with an enzyme having hydroxylaldehyde reductase activity encoded by the gene yqhD from E. coli, andthe microorganism is genetically modified for the improved production ofsaid hydroxy-2-keto-aliphatic acid metabolite by an increase of thehomoserine transaminase activity or the homoserine oxidase activityencoded by SerC from E. coli.
 2. The microorganism of claim 1, whereinthe aliphatic diol is ethylene-glycol and the hydroxy-2-keto-aliphaticacid metabolite is hydroxypyruvate.
 3. The microorganism of claim 1,wherein the aliphatic diol is 1,3-propanediol and thehydroxy-2-keto-aliphatic acid metabolite is 4-hydroxy-2-ketobutyrate. 4.The microorganism of claim 1, wherein the aliphatic diol is1,4-butanediol and the hydroxy-2-keto-aliphatic acid metabolite is5-hydroxy-2-ketopentanoate.
 5. A method for the fermentative productionof an aliphatic diol, comprising the steps of: culturing themicroorganism of claim 1 on an appropriate culture medium comprising asource of carbon and recovering the aliphatic diol from the culturemedium.
 6. The method of claim 5, wherein the diol is further purified.7. The method of claim 5, wherein the source of carbon is selected fromthe group consisting of: hexoses, pentoses, monosaccharides,disaccharides, oligosaccharides, molasses, starch and combinationsthereof.
 8. The method of claim 7, wherein the source of carbon isselected among glucose and sucrose.